Methods and systems for dissolution testing

ABSTRACT

Methods and systems for determining a dissolution profile of a sample material, and for solubilization screening of a library defined by an array comprising multiple sample materials are disclosed. The methods and systems are particularly advantageous for sampling and evaluation of very small samples, and can be advantageously applied in connection with evaluation of drug candidates.

RELATED APPLICATION

This application claims the benefit of and priority to co-owned U.S.provisional patent application Ser. No. 60/451,463 entitled “NovelMethods and Apparatus for Evaluating the Effects of Various Conditionson Drug Compositions Over Time” filed Mar. 1, 2003 by Carlson et al.

TECHNICAL FIELD

The present invention relates to the field of research for soluble formsof materials such as chemical compounds, with an emphasis in preferredembodiments on drug candidates. More particularly, the present inventionis directed toward methods and systems for rapidly evaluating thetime-dependent solubilization characteristics of materials such aschemical compounds (e.g., drug candidates). In particularly preferredembodiments, dissolution profiles are determined for libraries of samplematerials such as drug candidates, where such libraries are preparedusing high-throughput (i.e.,combinatorial) formulation protocols.

BACKGROUND OF THE INVENTION

This invention is directed at “dissolution” or “solubilization”, each ofwhich are used interchangeably herein to refer to a dynamicprocess—involving the kinetics by which a material dissolves into agiven media (e.g., solvent). Viewed from the perspective of theresulting solution, dissolution can be characterized by thetime-rate-of-change in concentration of the sample material in thesolution over a dissolution period. In contrast, “solubility” generallyrefers to an equilibrium condition (e.g., a thermodynamic value) andparticularly refers to how much of a sample material will dissolve in agiven medium under conditions in which thermodynamic equilibrium isachieved. In general, materials that have a high solubility willgenerally demonstrate faster dissolution than materials of lowersolubility. However, dissolution characteristics are not directly andspecifically correlatable to solubility, and valuable information aboutmaterials can be obtained by looking at dissolution profiles (e.g., inaddition to overall solubility data).

Dissolution testing of materials is typically practiced by dissolving atleast a portion of a material in a solvent to form a solution that has avarying concentration of the material over a dissolution period.Aliquots of the solution are then taken at various times during thedissolution period, and the concentration of the material in aliquot ismeasured. This information, taken collectively, represents atime-dependent dissolution profile. If allowed enough material andenough time to dissolve to reach saturation, one could also measuresolubility. However, a dissolution profile can be determined withoutnecessarily determining solubility. Dissolution testing is known in manyfields, but is of particular significance with respect to drugcandidates.

Combinatorial chemistry has revolutionized the process of drugdiscovery. See, for example, 29 Ace. Chem. Res. 1–170 (1996); 97 Chem.Rev. 349–509 20 (1997); S. Borman, Chem. Eng. News 43–62 (Feb. 24,1997); A. M. Thayer, Chem. Eng. News 57–64 (Feb. 12, 1996); N. Terret, 1Drug Discovery Today 402 (1996). Combinatorial chemistry has also beenapplied to materials research more generally. See, for example, U.S.Pat. No. 6,004,617 (generally), U.S. Pat. No. 5,985,356 (inorganicmaterials), U.S. Pat. No. 6,420,179 (organometallic materials), U.S.Pat. No. 6,346,290 (polymer materials), and U.S. Pat. No. 6,410,331(catalyst screening) to Schultz et al. See also U.S. Pat. No. 6,514,764(catalyst screening) to Willson.

Although combinatorial chemistry has to a great extent eliminated thebottleneck in early drug discovery, other bottlenecks have emerged ingetting a new drug to market. One such bottleneck is the identificationof a drug candidate that is soluble and/or that has an appropriate rateof solubilization in an aqueous solution such as water or bufferedwater. Low solubility and/or solubilization of a drug candidate can beproblematic because it can make the drug difficult to delivereffectively in a biological system. In fact, it has been estimated thatas many as thirty percent (30%) of drug candidates are discarded becausethey are poorly soluble (e.g., soluble to less then ten milligram permilliliter (<10 mg/ml)) and/or have poor solubilization. Drug candidatesare often sent back from animal toxicology and/or clinical trialsbecause of inability to formulate them into an acceptable delivery form(e.g., a soluble form, with appropriate solubilization characteristics).

Approaches to solve solubility and/or solubilization problems includeidentification of salt forms or related structures (e.g., polymorphs) ofthe drug candidate that may show equivalent activity and improvedsolubility and/or solubilization. However, such methods ofidentification (involving, for example, design, synthesis, andcharacterization of salt and polymorphic forms of a drug candidate) aregenerally time consuming, tedious and are by themselves bottlenecks ingetting a new drug to market.

Although efforts have been made to make certain aspects of suchapproaches more efficient (e.g., high-throughput investigation ofdrug-candidate polymorphs), the current state of the art has notadequately addressed a now-evident need for a high-throughput methodsand systems for determining solubilization characteristics of materialssuch as drug candidates. Of particular relevance, traditional methodsfor dissolution testing are further disadvantaged in that thedissolution screening of materials comprising drug candidate compoundsis generally done with a standard USP test equipment which requires alarge volume of fluid (e.g., 900 ml per sample) and a large amount ofsample (e.g., 100 mg to 200 mg). See, for example, U.S. Pat. No.4,924,716 to Schneider. The disadvantage of such known dissolutiontesting methods and systems is particularly evident in light of the factthat combinatorial synthesis methods generally result in new drugcandidate compounds being produced only in a limited quantity during theearly stage of discovery and/or lead optimization.

Accordingly, there is a need in the art to provide reliable,reproducible, high-throughput, dissolution screening methods and systemsthat only require a small amount of test sample.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide reliable,reproducible, high-throughput, dissolution screening methods and systemsthat only require a small amount of sample material for testing orscreening.

It is also an object of the invention to provide reliable systems forreproducibly sampling small amounts of materials.

Briefly, therefore, the present invention is generally directed tomethods for determining a dissolution profile for a sample material.

In one embodiment of such methods for determining a dissolution profile,not more than about 100 mg, preferably not more than about 50 mg, andmost preferably not more than about 20 mg of a sample material iscombined with a solvent. At least a portion of the sample material isdissolved in the solvent over a period of time to form a solution, withthe period of time defining a dissolution period. The solution has aconcentration of the sample material that varies during the dissolutionperiod. A portion of the solution is sampled successively in time atleast twice, and preferably at least three times during the dissolutionperiod. Specifically, the solution is sampled at a first time within thedissolution period to obtain a first aliquot of the solution, and at alater second time within the dissolution period to obtain a secondaliquot of the solution. The concentration of the sample material in thefirst aliquot of the solution, and the concentration of the samplematerial in the second aliquot of the solution is then determined.

In another embodiment of such methods for determining a dissolutionprofile, at least a portion of a sample material is dissolved in asolvent over a period of time to form a solution, with the period oftime defining a dissolution period. The solution has a concentration ofthe sample material that varies during the dissolution period. A portionof the solution is sampled successively in time at least twice, andpreferably at least three times during the dissolution period.Specifically, the solution is sampled at a first time within thedissolution period to obtain a first aliquot of the solution, and at alater second time within the dissolution period to obtain a secondaliquot of the solution. The difference between the first time and thesecond time is not more than about 2 minutes, preferably not more than 1minute. The concentration of the sample material in the first aliquot ofthe solution, and the concentration of the sample material in the secondaliquot of the solution is determined.

In a further embodiment of such methods for determining a dissolutionprofile, at least a portion of the sample material is dissolved in asolvent over a period of time to form a solution, with the period oftime defining a dissolution period. The solution has a concentration ofthe sample material that varies during the dissolution period. A portionof the solution is sampled at least twice, and preferably at least threetimes during the dissolution period. Specifically, the solution issampled at a first time within the dissolution period to obtain a firstaliquot of the solution, and at a later second time within thedissolution period to obtain a second aliquot of the solution. In thisembodiment, a portion of the first aliqout of the solution is furthersubsampled to obtain a first sub-aliquot of the solution, and then theconcentration of the sample material in the first sub-aliquot of thesolution is determined. Likewise, a portion of the second aliqout of thesolution is further subsampled to obtain a second sub-aliquot of thesolution, and

the concentration of the sample material in the second sub-aliquot ofthe solution is determined.

In preferred protocols of the embodiment in the immediately-precedingparagraph, the step of subsampling to obtain the first sub-aliquot ofthe solution results in a remainder portion of the first aliquot, andthe method further comprises returning the remainder portion of thefirst aliquot to the solution. In this case, the step of sampling at asecond time to obtain the second aliquot of the solution is effectedafter the remainder portion of the first aliquot is returned to thesolution. Also, the step of subsampling to obtain the second sub-aliquotof the solution results in a remainder portion of the second aliquot,and the method further comprises returning the remainder portion of thesecond aliquot to the solution.

In preferred protocols of the embodiment in the twoimmediately-preceding paragraphs, a first make-up aliquot of a liquidmedia is provided to the solution, where the first make-up aliquot has avolume about the same as the volume of the first sub-aliquot.Preferably, the first make-up aliquot is provided after sampling toobtain the first aliquot of the solution, and before sampling to obtainthe second aliquot of the solution. Likewise, a second make-up aliquotof a liquid media is provided to the solution, with the second make-upaliquot having a volume about the same as the volume of the secondsub-aliquot. Preferably, the second make-up aliquot being provided aftersampling to obtain the second aliquot of the solution.

The invention is also generally directed to methods for generating datadefining a dissolution profile for a sample material.

According to these methods, a dissolution profile is determinedaccording to any of the methods described in connection with theaforementioned embodiments, and then a first data point of thedissolution profile is defined by associating the determinedconcentration in the first aliquot (or first sub-aliquot) with the firsttime. Likewise, a second data point of the dissolution profile isdefined by associating the concentration determined in the secondaliquot (or sub-aliquot) with the second time. Preferably, suchassociation is effected using a microprocessor. The resulting data canbe a data set that is preferably displayed on a graphical user interfacein graphical or tabular form.

The invention is further generally directed to a system for determininga dissolution profile for a sample material.

The system comprises a sample container for dissolving at least aportion of the sample material in a solvent over a dissolution period oftime to form a solution that has a concentration of the sample materialthat varies during the dissolution period. The system also comprises anautomated sampling probe for sampling a portion of the solution at leasttwice during the dissolution period, the solution being sampled at firstand second times within the dissolution period to obtain first andsecond aliquots of the solution, respectively. The sampling probe has adistal end positionable in fluid communication with the solution in thesample container, and has a proximate end, that is opposing the distalend and in fluid communication therewith. The sampling probe has one ormore fluid cavities and can comprise conduits integrally formed in aunitary body or supported by the probe body for providing fluidcommunication between the distal end and the proximate end of thesampling probe. The system also comprises a sub-sampling device, whichis preferably a sampling valve, in fluid communication with theproximate end of the sampling probe. The sub-sampling device isconfigured for subsampling a portion of each of the first and secondaliquots of the solution to obtain first and second sub-aliquots of thesolution. The system comprises as well an analytical unit fordetermining the concentration of the sample material in the first andsecond sub-aliquots of the solution. In another embodiment, the systemfurther comprises an automated dispensing probe for providing the samplematerial to the container and/or for providing a make-up aliquot to thecontainer. The dispensing probe and sampling probes are preferably underseparate functional control from each other (allowing for independentdispensing and/or sampling therefrom), but may be structurallyintegrated through a common probe head, allowing for integratedpositional control of the dispensing probe and the sampling probe.

The invention is also generally directed to a system for automatedsampling of small volume liquid samples.

This system comprises a sample container for containing a liquid sample,and an automated liquid handing system comprising a sampling probe, arobotic arm for translating the sampling probe, and a pump in continuousor selectable fluid communication with the sampling probe for providinga motive force at least for withdrawing a portion of the liquid sampleinto the probe to effect sampling. The sampling probe has a distal endand a proximate end, with the distal end of the sample probe beingpositionable in fluid communication with the liquid sample in the samplecontainer for sampling a portion of the liquid sample to obtain asampled aliquot, and with the proximate end being opposing the distalend and in fluid communication therewith. The sampling probe has one ormore fluid cavities and can comprise conduits integrally formed in aunitary probe body or supported by the probe body for providing fluidcommunication between the distal end and the proximate end of thesampling probe. A sampling valve provides fluid communication betweenthe proximate end of the sampling probe and the pump. The sampling valveis a multi-port sampling valve comprising a sample loop, and thesampling valve is configured in at least a first selectable position anda second selectable position. The sampling valve is configured forloading at least part of the sampled aliquot into the sample loopthrough a sampling flow path from the proximate end of sampling probe tothe first pump. The sampling valve is further configured for dischargingthe contents the sample loop from the sampling valve as a sub-aliquot ofthe sampled aliquot. In another embodiment, this system furthercomprises an automated dispensing probe for providing the samplematerial to the container and/or for providing a make-up aliquot to thecontainer. The dispensing probe and sampling probes are preferably underseparate functional control from each other (allowing for independentdispensing and/or sampling therefrom), but may be structurallyintegrated through a common probe head, allowing for integratedpositional control of the dispensing probe and the sampling probe.

These inventions provide several advantages, particularly in connectionwith sampling of, and dissolution profiling of small amounts samplematerials. These inventions are useful in many fields, including withoutlimitation in characterization of materials in high-throughput orcombinatorial workflows. In a preferred application, the inventions canbe advantageously applied for evaluating dissolution characteristics ofdrug candidates or drug compositions.

Other features, objects and advantages of the present invention will bein part apparent to those skilled in art and in part pointed outhereinafter. All references cited in the instant specification areincorporated by reference for all purposes. Moreover, as the patent andnon-patent literature relating to the subject matter disclosed and/orclaimed herein is substantial, many relevant references are available toa skilled artisan that will provide further instruction with respect tosuch subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic of an illustrative high throughput solubilityscreening system in accordance with the principles of the presentinvention;

FIG. 2 shows a side view of a dispensing unit in accordance with theprinciples of the present invention;

FIG. 3 shows a front view of a dispensing unit with a controlledagitation assembly in accordance with the principles of the presentinvention;

FIG. 4 shows a schematic of an illustrative analytical unit inaccordance with the principles of the present invention;

FIGS. 5A and 5B show schematics of illustrative multi-port samplingvalves in accordance with principles of the present invention, includinga six-port, single sample loop configuration (FIG. 5A) and a ten-port,two sample loop configuration (FIG. 5B);

FIG. 6 shows a schematic of another illustrative multi-port samplingvalve in accordance with principles of the present invention, includinga ten-port, two sample loop configuration adapted to allow for providinga make-up aliquot of a liquid media to the solution being evaluated;

FIG. 7 shows a dissolution profile of Aspirin obtained from theprotocols of Example 1;

FIG. 8 shows a schematic of an illustrative library design of sampleformulations used in Example 2.

FIG. 9 shows a schematic of illustrative images of the library of sampleformulations that could be obtained from a visual inspection screeningused in Example 2;

FIG. 10 shows a schematic of illustrative volume average particle sizedistributions of the selected formulations used in Example 2;

FIG. 11 shows a schematic of illustrative diffraction patterns and anillustrative table of corresponding calculated crystallinity for theselected formulations used in Example 2; and

FIG. 12 shows a schematic of illustrative dissolution profiles anddissolution times of the selected formulations used in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The invention as described and claimed herein generally includes methodsfor determining a dissolution profile of a sample material or ofmultiple sample materials that are members of an array or library. Theinvention also includes methods for generating data, such as a data set,for defining a dissolution profile of a sample material or of multiplesample materials that are members of an array or library. The inventionincludes as well, a system for determining a dissolution profile of asample material or of multiple sample materials that are members of anarray or library. In another aspect, the invention more generallyincludes a system for automated sampling of small volume liquid samples,or of multiple liquid samples that are members of an array or library.The particular nature of each of these aspects of the invention aredescribed hereinafter.

Methods for Determining a Dissolution Profile

One aspect of the invention is directed toward methods for determining adissolution profile of a sample material, or of multiple samplematerials that are members of an array or library.

These methods generally comprise (i) dissolving at least a portion ofthe sample material in the solvent over a period of time to form asolution, where the period of time defines a dissolution period, and thesolution has a concentration of the sample material that varies duringthe dissolution period, (ii) sampling a portion of the solution at leasttwice during the dissolution period, the solution being sampled at afirst time within the dissolution period to obtain a first aliquot ofthe solution, and at a later second time within the dissolution periodto obtain a second aliquot of the solution, and (iii) analyzing thefirst and second aliquots of the solution to determine the concentrationof the sample material in the first aliquot of the solution, and todetermine the concentration of the sample material in the second aliquotof the solution. The dissolving is preferably initiated by, and thesemethods can generally further comprise, (iv) combining an amount of thesample material with an amount of a liquid media (e.g., a solvent) intowhich the sample material will dissolve. In preferred embodiments, thesolution is preferably sampled at least three times during thedissolution period, such that the method as described further comprisessampling a portion of the solution at a later third time within thedissolution period to obtain a third aliquot of the solution, andanalyzing the third aliquot to determine the concentration of the samplematerial in the third aliquot of the solution.

These methods are particularly characterized by one or more of thefollowing features (A–M), considered alone or in combination in each andevery possible permutation. Generally considered, the characterizingfeatures of the invention include:

-   -   (A) the type of sample material (e.g. drug candidate);    -   (B) the relatively small amount (e.g. weight) of the sample        material (e.g., not more than about 50 mg);    -   (C) the relatively small amount (e.g., volume) of the media        (e.g., solvent) with which the sample material is combined to        initiate dissolution (e.g., not more than about 50 ml);    -   (D) the protocols (including specific methods and/or devices) by        which dissolution is initiated and initial sampling is effected        (e.g., sampling the first aliquot from the solution within a        very short period of time, such as not more than about 1 minute,        after initiation of dissolution);    -   (E) the protocols (including specific methods and/or devices) by        which sampling is effected to obtain an accurate, small volume        sample (e.g., sampling at least a portion of the solution to        obtain an aliquot of the solution, and then subsampling a        portion of the sampled aliquot to obtain sub-aliquot of the        solution), where the sub-aliquot can then be analyzed to        determine the concentration of the sample material in the        solution;    -   (F) the protocols (including specific methods and/or devices) by        which the amount of solution being sampled is preserved to allow        for multiple samples even from a relatively small starting        volume (e.g., by, in addition to sampling and subsampling        protocols as described above, the combination of returning the        remainder portion of the aliquot (the portion of the aliquot not        included within the sub-aliquot) to the solution (e.g. to the        container holding the solution);    -   (G) the protocols (including specific methods and/or devices) by        which the total volume of the solution being sampled is        substantially preserved to allow for more direct comparison of        determined concentration values for multiple samples (e.g., by,        independently, or in addition to the sampling and subsampling        and/or remainder-returning protocols as described above,        providing a make-up aliquot of a liquid media (e.g. the solvent)        to the solution);    -   (H) the protocols (including specific methods and/or devices)        for maintaining a dispersion formed by combining a solid sample        material with a solvent, the dispersion comprising the solution        being sampled and the undissolved solid sample material        dispersed in the solution (e.g., through agitation); and    -   (I) the protocols (including specific methods and/or devices)        for sampling the solution of a dispersion, while allowing the        undissolved sample material to remain in the dispersion (rather        than being sampled off with the solution) (e.g., filtering of        the sampled aliquots during or after sampling, preferably with        back-flushing of the filter media so that undissolved solid        sample material remains exposed to the solvent for continued        dissolution into the solution).    -   (J) the relatively fast sampling frequency defined by the        difference in time between sampling of successive aliquots        (e.g., the difference between the first time and the later        second time at which the first aliquot and the second aliquot        are sampled, respectively) (e.g., the sampling frequency being        not more than about 1 minute);    -   (K) the protocols (including specific methods and/or devices)        that define a sampling plan for efficient sampling, particularly        for efficiently sampling multiple members of an array of sample        materials (e.g., a sampling plan that includes for each sample,        sampling the solution at least six times over at least two        distinct sampling intervals, including a first sampling interval        proximate to the initiation of dissolution that involves        sampling the solution at least three times, and a later-in-time        second sampling interval separated substantially from the first        sampling interval that also involves sampling the solution at        least three times);    -   (L) the protocols (including specific methods and/or devices) by        which these methods can be used to obtain a dissolution profile        that is representative of dissolution in a dynamic environment,        such as would mimic the environment along various regions of the        gastrointestinal tract (e.g., by varying, and preferably        controllably varying, a property, such as pH, of the solvent        over time);    -   (M) the protocols (including specific methods and/or devices) by        which these methods can be applied to a high-throughput method        for evaluating a library or an array of sample materials, (e.g.,        an array comprising multiple members of sample materials in        containers formed on or supported by a common substrate);        Because the invention is contemplated and is defined with        respect to each and every possible combination and permutation        of these characterizing features (A–M), a person of skill in the        art would readily appreciate, for example, that these methods of        the invention can be defined by the above-recited general steps        (i), (ii) and (iii), and optionally (iv), in combination with        one or more of features A–M. As a non-limiting example, these        methods of the invention can be characterized by the combination        feature A plus any one or more of the features B, C, D, E, F, G,        H, I, J, K, L and M, the combination of feature B plus any one        or more of features C, D, E, F, G, H, I, J, K, L and M, the        combination of feature C plus any one or more of features D, E,        F, G, H, I, J, K, L and M, the combination of feature D plus any        one or more of features E, F, G, H, I, J, K, L and M, the        combination of feature E plus any one or more of features F, G,        H, I, J, K, L and M, the combination of feature F plus any one        or more of features G, H, I, J, K, L and M, the combination of        feature G plus any one or more of features H, I, J, K, L and M,        the combination of feature H plus any one or more of features I,        J, K, L and M, the combination of feature I plus any one or more        of features J, K, L and M, the combination of feature J plus any        one or more of features K, L and M, the combination of feature K        with any one or more of features L and M, and the combination of        feature L with feature M.

In particularly preferred embodiments, the invention is defined by thegeneral steps characterized by many of the features A-M in combination,including for example features B, D, E, F and J together in combinationwith each other, and optionally in further combination with one or moreof the other features A, C, G, H, I, K, L and M.

Methods for Generating Data for Defining a Dissolution Profile

Another aspect of the invention includes methods for generating data,such as a data set, for defining a dissolution profile of a samplematerial or of multiple sample materials that are members of an array orlibrary.

Generally, such methods comprise the general steps (i), (ii) and (iii),and optionally (iv) as described above in connection with the methodsfor determining a dissolution profile of a sample material (or ofmultiple sample materials that are members of an array or library), assupplemented herein. These methods further comprise associating thedetermined concentration of the sample material in particular aliquotswith the particular respective times at which such aliquots were taken(e.g., associating the determined concentration of the sample materialin the first aliquot with the first time to define a first data point ofthe dissolution profile, and associating the determined concentration ofthe sample material in the second aliquot with the second time to definea second data point of the dissolution profile). Preferably, suchassociation is implemented with a microprocessor such as a personalcomputer. Preferably, the generated data, such as a data set, is storedin a memory device. Preferably, the generated data, such as a data set,can be retrieved from memory, for example, for being displayed on agraphical user interface.

Additionally, these methods are also particularly characterized by oneor more of the aforementioned features (A–M), considered alone or incombination in each and every possible permutation, with preferredcombinations and permutations as described above.

System for Determining a Dissolution Profile

In another aspect, the invention includes systems for determining adissolution profile of a sample material or of multiple sample materialsthat are members of an array or library.

These systems generally comprise (i) a sample container for dissolvingat least a portion of the sample material in a solvent over adissolution period of time to form a solution that has a concentrationof the sample material that varies during the dissolution period, (ii)an automated sampling probe for sampling a portion of the solution atleast twice during the dissolution period, the solution being sampled atfirst and second times within the dissolution period to obtain first andsecond aliquots of the solution, respectively, and (iii) an analyticalunit for determining the concentration of the sample material in thefirst and second sub-aliquots of the solution. Generally, such systemspreferably also comprise a microprocessor, most preferably together withautomation software for controlling the sampling probe, and a controlsystem and software for controlling the analytical unit.

Additionally, these systems are also particularly characterized by oneor more of features A and B (with respect to defining a fill level of acontainer or a container maximum fill volume), and/or by one or more ofthe features D, E, F, G, H and I, as described generally above and inmore detail below. In a specific, non-limiting example, the samplingprobe can comprise a distal end positionable in fluid communication withthe solution in the sample container, and a proximate end in fluidcommunication with a subsampling device, in which the sub-samplingdevice is configured for subsampling a portion of each of the first andsecond aliquots of the solution to obtain first and second sub-aliquotsof the solution. These systems are further particularly characterized bya control system (including software) effective for implementing one ormore of the aforementioned features J and K, L and M. Suchcharacterizing features form a defining part of the systems of theinvention considered alone or in combination in each and every possiblepermutation, with preferred combinations and permutations as describedabove.

Systems for Automated Sampling of Small Volume Liquid Samples

A further aspect of the invention involves systems for automatedsampling of small-volume liquid samples, or of multiple liquid samplesthat are members of an array or library.

These systems generally comprise (i) a sample container for containing aliquid sample, (ii) an automated liquid handing system comprising asampling probe, a robotic arm for translating the sampling probe, and apump in fluid communication with the sampling probe for providing amotive force at least for withdrawing a portion of the liquid sampleinto the probe to effect sampling, where the sampling probe has a distalend and a proximate end, the distal end of the sample probe beingpositionable in fluid communication with the liquid sample in the samplecontainer for sampling a portion of the liquid sample to obtain ansampled aliquot, and (iii) a sampling valve providing fluidcommunication between the proximate end of the sampling probe and thepump, where the sampling valve is a multi-port sampling valve comprisinga sample loop, the sampling valve is configured for loading at leastpart of the sampled aliquot into the sample loop through a sampling flowpath from the proximate end of sampling probe to the first pump, and thesampling valve is further configured for discharging the contents thesample loop from the sampling valve as a sub-aliquot of the sampledaliquot.

Additionally, these systems are also particularly characterized by oneor more of features A and B (with respect to defining a fill level of acontainer or a container maximum fill volume), and/or by one or more ofthe features D, E, F, G, H and I, as described generally above and inmore detail below. These systems are further particularly characterizedby a control system (including software) effective for implementing oneor more of the aforementioned features J and K, L and M. Suchcharacterizing features form a defining part of the systems of theinvention considered alone or in combination in each and every possiblepermutation, with preferred combinations and permutations as describedabove.

Such systems can be employed in a number of applications for which it isdesirable to enjoy the advantages of the present invention, especiallyin connection with automated sampling of small-volume liquid samples, orof multiple small-volume liquid samples that are members of an array orlibrary. For example, one could apply this system for evaluatingcompound stability and/or excipient compatability of drug candidates,especially in drug compositions. In a preferred such application that isan alternative to dissolution testing, such systems can be employed inscreening systems such as those described in co-owned, co-pendingapplication U.S. Serial No. 60/451,463 entitled “Novel Methods andApparatus for Evaluating the Effects of Various Conditions on DrugCompositions Over Time” filed Mar. 1, 2003 by Carlson et al., which ishereby incorporated by reference for all purposes.

Further Characterizing Features

More specific aspects of the various steps of the methods and of variouscomponents of the systems, and in particular, more specific aspects ofeach of the aforedescribed characterizing features (A–M) are describedhereinafter. Such description includes more specific protocols(including specific methods and/or devices) for effecting such steps andfor realizing such features. Since each of these more specific aspectsmore specifically define the general features outlined above, each ofthese more specific aspects are also characterizing features of themethods and the systems of the invention, considered alone or incombination in each and every possible permutation.

Sample Materials (Feature A)

As used herein, a sample refers to a single, discrete, individual unitof a material that is being evaluated. The sample material is anelement, compound or composition being evaluated or tested or screened,for which a time variation in a property thereof is being determined,including for example in a preferred application, for which thedissolution profile is to be determined. The sample material cancomprise or consist essentially of an organic material, an inorganicmaterial, an organometallic material, or a combination thereof. Thesample material can comprise or consist essentially of a polymer (e.g. abiological polymer or a non-biological polymer), or a compositematerial.

Preferred organic materials include small organic compounds (e.g.pharmaceuticals, agrichemicals, etc.) or biological polymers (e.g.,nucleic acid polymers such as oligonucleotides, deoxyribonucleic acidpolymers (DNA), ribonucleic acid polymers (RNA), etc., and amino acidpolymers such as peptides, proteins, enzymes, etc.).

In some preferred embodiments, the sample material is a drug sample. Adrug sample refers to a sample that is a drug candidate, a combinationof drug candidates, or a drug composition. A drug composition refers toa composition that has one or more drug candidates and at least oneexcipient. Hence, the sample material preferably comprises or consistsessentially of a drug candidate. A drug candidate is a compound (salt orneutral) shown under one or more various assays to have pharmacological(prophylactic or therapeutic) activity. A drug candidate may also be,but has not necessarily been, also shown to be safe under varioustoxicity assays. An active pharmaceutical ingredient (API) is a specificcompound (salt or neutral), typically that has been approved by agovernmental entity for use in a pharmaceutical, e.g., has beendemonstrated to be, and is typically approved by a governmental entity(e.g., U.S. Food and Drug Administration) to be safe and effective for aparticular indication. The methods and systems described in this patentapplication are preferred for use in evaluating sample materials thatare drug candidates, which may or may hot be APLs. And, as those ofskill in the art will appreciate, the exact API or drug candidate is notcritical to this invention, but is typically a small organic molecule.In some cases, the drug candidate or API can be a biological polymersuch as an oligonucleotide, a DNA, a RNA, a cDNA, a polypeptide or aprotein. Some drug candidates have salts that are anionic or cationicand some drug candidates are neutrals. No matter the form, drugcandidates may have different crystallographic polymorphs. Herein, theterm polymorph is intended to include polymorphs, pseudo-polymorphs,hydrates, solvates and the like. The term excipient refers to a drugcomposition component that is typically intended to aid in manufacture,administration, absorption, appearance enhancement or retention ofquality of a drug. Excipients rarely, if ever, possess pharmacologicalactivity by themselves, and are accordingly loosely characterized asbeing substantially “inert.” However, excipients can initiate, propagateor participate in chemical or physical interactions with a drugcandidate, possibly leading to compromised or enhanced quality orperformance of the drug. One example of an excipient that is commonlyused is a solvent. For example, solvents may have an effect on thereaction rate of a drug candidate, or the degradation rate of a drug maychange with the dielectric constant of the medium. As a specificexample, certain studies have shown an increase in photo stability ofVitamin-B12 by the addition of viscogens such as glycerol or Ficoll. SeeRong, Lui (editor), Water-insoluble Drug Formulation, Chapter 7“Solubilizatinon Using CoSolvent Approach”, J. Trivedi and M. Wells(authors), Interpharm Press, 2000, pp. 141–168, which is herebyincorporated by reference. One or more excipients used together with oneor more drug candidates in a drug composition, can have a significanteffect on dissolution of a drug composition sample material, andtherefore, on the bioavailability of such drug sample.

The particularly physical state or form of the sample material is notnarrowly critical to the invention. The sample material, such as a drugcandidate or a drug composition, can take any form, such as a liquid, asolid, a gel, and the like. In preferred embodiments, the samplematerial is in the form of a solid, such as a crystalline solid.Crystalline solids can be single crystals or polycrystalline. The samplematerial can, in some embodiments, be provided for evaluation already ina partially dissolved state, including for example, as a suspension ordispersion (including both uniform and non-uniform dispersions), or as asolid-liquid emulsion. A dispersion, for example, can comprise apartially dissolved solid sample material dispersed in a solution—thatis, dispersed in a liquid media into which the solid sample material isdissolving. In preferred embodiments, the sample material is provided asa substantially uniform dispersion, or is provided as a solid andcombined with a solvent to form a substantially uniform dispersion.

Amount of Sample Materials (Feature B)

The amount of sample material being evaluated or tested or screened isnot critical to many embodiments of the invention, but as noted above,the invention offers particular advantages with respect to methods andsystems for evaluating or testing or screening relatively small amounts(e.g. weight) of sample materials. Hence, in preferred embodiments, theamount of sample material provided to a sample container is preferablynot more than about 100 mg, more preferably not more than about 50 mg,and most preferably not more than about 20 mg. Generally, the amount ofsample material can also be characterized as being not more than about10 mg, not more than about 5 mg, not more than about 2 mg, or not morethan about 1 mg. Also, for some applications, such as for combinatorial(high-throughput) screening of formulations such as drug compositions,the amount of sample material can be characterized as being not morethan about 500 ug (0.5 mg), or not more than about 100 ug (0.1 mg), ornot more than about 10 ug (0.01 mg) or not more than about 1 ug (0.001mg). As used in the context of the amount of sample material, the term“about” refers to a degree of error associated with the type ofinstrument (e.g. weighing scale) used to determine the amount, based ona statistically significant and statistically acceptable basis fordetermining such error.

Solvent/Amount of Solvent (Feature C)

The solvent generally refers to the liquid media into which the samplematerial is dissolved during the dissolution test. The particular typeof liquid media used in connection with the methods of this invention isnot narrowly critical, and can be selected based on the type of samplematerial being investigated. Generally, the liquid media can be asolvent having a single unitary chemical composition (e.g.,substantially pure solvents consisting essentially of one type ofsolvent, such as water, ethanol, etc.) or can be a solvent having acombination of chemical compositions (e.g., co-solvents comprising twoor more miscible or imiscible solvents, such as water/ethanolco-solvents). The solvent can generally include one or more componentsor agents for controlling ionic strength (e.g., different salts orconcentrations of salts), pH, pOH, or one or more agents or componentsthat are solubilizers, disintegrants, or surfactants.

In some preferred applications, such as applications for evaluatingdissolution of drug candidates, the liquid media can be water or anaqueous media, such as a buffered aqueous media, including withoutlimitation buffered solutions having a pH ranging from about 2 to about10, preferably from about 2 to about 4, from about 3 to about 5, fromabout 4 to about 7, from about 6 to about 8, from about 6 to about 9, orfrom about 7 to about 10. The liquid media used as the solvent can alsobe biological fluids (e.g., blood, saliva, gastric fluid), or mimics ofsuch fluids (e.g., artificial blood or artificial saliva, etc.).

The amount of solvent combined with the sample material in the samplecontainer for evaluation is not critical to many embodiments of theinvention. Generally, the sample material is combined with an amount ofsolvent effective for forming a solution having a detectableconcentration of sample material in the solution. As such, a person ofskill in the art will appreciate that the amount of solvent to becombined with the sample material can be determined based on the type ofsample material, the amount of the sample material, and the sensitivityof the analytical unit or system. In one general approach, the amount ofsolvent can be controlled to obtain a concentration of the samplematerial in solution that provides for non-equilibrium (e.g., withrespect to dissolution, non-saturation) conditions (sometimes referredto as “sink” conditions) during the evaluation period. For example, theamount of solvent can be controlled to obtain a concentration of thesample material in solution that is within the range of about 5% toabout 25% of the equilibrium solubility, preferably from about 10% toabout 20% of the equilibrium solubility, in each case of that samplematerial in that solvent at the test temperature of interest.

In some preferred applications, such as applications for evaluatingdissolution of drug candidates, the sample material is combined with anamount of solvent effective for forming a solution having aconcentration of sample material in the solution ranging from about 0.01mg/ml to about 10 mg/ml, preferably from about 0.1 mg/ml to about 5mg/ml. As a non-limiting example, an amount of sample material rangingfrom 0.01 mg to 0.5 mg is combined with an amount of solvent rangingfrom 1 ml to 10 ml. As further non-limiting examples, the samplematerial (e.g., a drug candidate-containing sample) can be combined witha solvent with the following relative amounts: not more than about 100mg of sample material with not more than about 100 ml of solvent; notmore than about 50 mg of sample material with not more than about 50 mlof solvent; not more than about 20 mg of sample material with not morethan about 20 ml of solvent; not more than about 10 mg of samplematerial with not more than about 10 ml of solvent; not more than about5 mg of sample material with not more than about 5 ml of solvent; notmore than about 2 mg of sample material with not more than about 2 ml ofsolvent; not more than about 1 mg of sample material with not more thanabout 1 ml of solvent; not more than about 0.1 mg of sample materialwith not more than about 0.1 ml of solvent; and not more than about 0.01mg of sample material with not more than about 0.01 ml of solvent.

As noted above, the invention offers particular advantages with respectto methods and systems for evaluating or testing or screening relativelysmall amounts (e.g. weight) of sample materials, and also for evaluatingsuch sample materials in relative small volumes of solution. Hence, inpreferred embodiments, the total amount of solvent to be combined withthe sample material in a sample container (and substantiallycorrespondingly, the total amount of the resulting solution—e.g., of adispersion comprising the solution and undissolved solid sample materialthat is formed from the combination of a solvent and a solid samplematerial) is preferably not more than about 100 ml, more preferably notmore than about 50 ml, and most preferably not more than 20 ml. In manyapplications, it will be preferred to be a total solution volume of notmore than about 10 ml, or not more than about 5 ml, or not more thanabout 2 ml, or not more than about 1 ml, or not more than about 0.5 mlor not more than about 0.1 ml. Preferably, such total amount of solventranges from about 0.1 ml to about 50 ml, preferably from about 0.5 ml toabout 20 ml, and most preferably from about 1 ml to about 10 ml or fromabout 2 ml to about 5 ml.

As used in the context of the amount of solvent or the amount of totalsolution, the term “about” refers to a degree of error associated withthe type of instrument (e.g. volumetric measure) used to determine theamount, based on a statistically significant and statisticallyacceptable basis for determining such error.

Initiation of Dissolution and Initial Sampling (Feature D)

Dissolution is initiated by combining the sample material a solvent,preferably with a suitable solvent, and preferably under conditions(e.g., temperature) effective for causing at least a portion of thesample material to dissolve in the solvent. The sample material andsolvent can be combined in a container such as a sample container. Theorder of addition is not critical to the invention, and can includeadding the solvent to the contained sample material, or adding thesample material to the contained solvent. In some preferred approaches,the sample material is provided to the container first, and then thesolvent is added to the contained sample material.

The type of container is not critical to the invention. The containercan be an individual container, such as a vial, beaker, test-tube,flask, etc. For many applications, including for example forcombinatorial or high-throughput applications (described more fullybelow), an array of multiple sample materials are contained in multiplecontainers, respectively, with each sample material in its own discrete,dedicated container. In such cases, the containers can be structurallyintegrated, such as being formed in or supported by a common substrate.Hence, for example, the containers can be wells of a microtiter plate,or can be individual vials supported in wells of a microtiter plate.Standard microtiter formats (typically having a 0.9 mm center-to-centerdistance between adjacent wells or vials—that is, a 0.9 mm “pitch”), andtypically in an 8 row by 12 column (or vice-versa) format, that is an“8×12 format”, is particular useful for performing high throughputreaction and screening of samples. Another preferred format for slightlylarger volume containers is a ninety-six well plate in an 8×12 formatwith about a 20 mm pitch (center-to-center distance of adjacent wells orvials). Other microtiter formats can also be employed, including384-well plates. In general, the number of containers (e.g., vials orwells) can be 96×N, where N ranges from 1 to about 20, and preferablyfrom 1 to 5. The containers can be open during the dissolution process,or can be sealed, such as hermetically sealed, during the dissolutionprocess, and can be isolated from each other (e.g., to avoidcross-contamination between adjacent containers). The containers can beof any suitable material, including for example glass, plastic, metal,etc. Metals such as aluminum are preferred in some embodiments (e.g.,for control of thermal properties of the solution).

The sample material is combined with the solvent at an initiation time,t_(o), to initiate dissolution of the sample material in the solvent. Inpreferred protocols, the dissolution profile is characterized very earlyin the dissolution period—to provide valuable information about thekinetics of the initial part of the dissolution process. Accordingly,the first and second aliquots of the solution are preferably sampled assoon as practical after the initiation of dissolution, and particularly,within the initial interval of time that includes the fastestdissolution rate (i.e., the highest time-rate-of-change of concentrationof the sample material in the solution). In preferred embodiments, thesolution is sampled at least three times during the dissolution period,with each of the first, second and third aliquots being sampled as soonas practical after the initiation time, and particularly, within theinterval of time that includes the fastest dissolution rate.

More specifically, in some applications, the first time at which thesolution is sampled to obtain the first aliquot is within about 1 minuteafter the initiation time, t_(o). Preferably in some cases, the firsttime at which the solution is sampled to obtain the first aliquot iswithin about 30 seconds after the initiation time, t_(o), within about10 seconds after the initiation time, t_(o), or within about 5 secondsafter the initiation time, t_(o), or within about 2 seconds after theinitiation time, t_(o). The sampling frequency for the subsequent secondsampling, defined by the difference in time between the first time andthe second time, is preferably not more than about 1 minute. Likewise,where a subsequent third sample is done to obtain a third aliquot of thesolution as part of the initial set of sampling, the sampling frequencyfor the third sampling, defined by the difference in time between thesecond time and the third time, is also preferably not more than about 1minute. Hence, it can be appreciated that the a first sampling intervalcomprising the first time, the second time, and preferably also thethird time is preferably initiated directly after and proximate to theinitiation time, and at any rate, the first sampling interval can becompleted with two samples in not more than about 2 minutes total, andwith three samples with not more than about 3 minutes total, to obtainkinetic information about the onset of dissolution. More specificallypreferred sampling frequencies are discussed below (in connection withFeature J), and such preferred sampling frequencies are particularlyapplicable to the present discussion as well.

The above description details the methodologies by which dissolution isinitiated, by which initial sampling is effected (e.g., sampling thefirst aliquot from the solution) within a very short period of timeafter initiation of dissolution, and by which a dissolution profile isobtained for a first sampling interval that is proximate in time to theinitiation of dissolution. Such methodologies can be advantageouslyeffected using the system described below in connection with thepreferred embodiments of the invention.

In particular, such methodologies can be effected using, in combination,an automated dispensing probe for providing either or both of the samplematerial and/or the solvent to the container (e.g., at the initiationtime, t_(o)), and an automated sampling probe for sampling the solutionto obtain the first and second aliquots of the solution during thedissolution period.

The automated dispensing probe can be a component of an automated solid(e.g., powder) dispenser, such as the Powderinium (Autodose, S.A.,Geneva, Switzerland) or of an automated liquid dispenser, such as theCavro (Tecan Scientific Instruments, San Jose, Calif.). Automated soliddispensers can further comprise a robotic arm for translating theautomated dispensing probe for positioning, a hopper or other sourcecontainer for the material to be dispensed, and automation software forcontrolling the dispenser. Automated liquid dispenser can be anautomated liquid handing robot that further comprises a robotic arm fortranslating the automated dispensing probe for positioning, a sourcecontainer for the solvent or sample material to be dispensed, a pump influid communication with the dispensing probe for providing a motiveforce for dispensing, for example the solvent, into the container, andautomation software for controlling the dispenser.

The automated sampling probe can have a distal end positionable in fluidcommunication with the solution in the container for sampling thesolution during the dissolution period. The automated sampling probe ispreferably a component of an automated liquid handing unit or system,with the liquid-handling unit or system further comprising a robotic armfor translating the automated sampling probe for positioning, a pump influid communication with the sampling probe for providing a motive forcefor withdrawing a portion of the solution into the probe to effectsampling, and automation software for controlling the sampling probe.

The dispensing probe and the sampling probe can be structurallyindependent of each other, or can be structurally integrated with eachother (e.g., using a common probe head that supports each of thedispensing probe and the sampling probe). In either case, the functionof the dispensing probe and the function of the sampling probe cancontrolled independently of each other. That is, the functionality of,and the control of the functionality of, at least the dispensingfunction of the dispensing probe and the sampling function of thesampling probe is independent of each other, such that dispensing ofsample material or solvent can occur at the same time, or in closetemporal proximity to sampling to obtain the first aliquot of thesolution. The independence of the control goes towards being able toeffectively coordinate the timing of such functionality, and does notnecessarily imply that there is no control communication between the twodevices. For example, it may be desirable to haveindependently-controlled devices in which the control signal forinitiating the initial sampling with the sampling probe is triggered bya signal from the dispensing probe indicating that the dispensing hasbegun (and that therefore, dissolution has been initiated). Further, theautomation software for these devices is can be separate, or can bepartially or totally integrated.

Sampling, Subsampling and Returning of Remainder Portion (Features E, F)

In a preferred protocol, a portion of each of the sampled aliquots ofthe solution (e.g., the first aliquot and the second aliquot) aresubsampled to obtain corresponding sub-aliquots (e.g., a firstsub-aliquot from the first aliquot and a second sub-aliquot from thesecond aliquot) of the solution. The subsampling refers to a furthersampling of a portion of the original sample withdrawn from the solutionin the container (the original sampled aliquot) to obtain a sub-aliquotthat represents a smaller portion (e.g., smaller volume) than the volumeof the original aliquot withdrawn from the container. Such subsamplingoffers several advantages over a direct sampling approach in which avery small volume is withdrawn in the first instance directly from thecontainer. First, such subsampling allows for a larger initial samplesize to be withdrawn, which may be more representative of the bulk orphysically-averaged properties (e.g. concentration) of the solution thanthat of a locally drawn very small volume sample. Also, the ability todo the original sampling in a larger volume size facilitates theengineering of the automated sampling and other processes such asfiltering, so that macroscale devices such as sampling lines, pumps(e.g. syringe pumps), valves, filters, seals can be employed.Significantly, such subsampling can affords improved reliability andreproducibility of the size of the subsampled sub-aliquot, which can beimportant in providing a comparative basis. The subsampling approachalso improves precision and sensitivity of analysis, since in preferredembodiments, it allows for on-line combination of sampling and analysis(i.e., direct fluid communication between the sampling unit and theanalytical unit) without the need for further handling of subsampledsub-aliquot.

The sub-aliquot can be of any volume suitable for detection, but cangenerally be as small as less than about 100 μl, preferably less thanabout 50 μl, and in some cases, less than about 20 ul, less than about10 ul, less than about 5 ul, or less than about 1 ul.

In a further preferred protocol that is related to the subsamplingprotocol described in the immediately-preceding paragraphs, the methodcan further comprise, after subsampling a sampled aliquot to obtain asub-aliquot of the solution, the step of then returning a remainderportion of the sampled aliquot to the solution, the remainder portionbeing the portion of the orginally-sampled aliquot that was not includedwithin the sub-aliquot obtained by subsampling. Advantageously,returning the remainder portion from each of the subsampled aliquots tothe solution has the net effect of minimizing the amount of solutionthat is ultimately permanently taken from the solution. That is, the netdepletion of solution is only the amount of the sub-aliquot obtained bysub-sampling. Another significant advantage of returning the remainderportion to the solution relates to a particularly-preferred embodimentin which the sampling path for the original sampling is physically thesame as the remainder-return flow path, but with the fluid directionreversed relative to the forward, sampling flow path. In thisembodiment, for example, an in-line filter can be used in the flow pathallowing for filtering of the sampled aliquot during withdrawal of thealiquot in the sampling path (forward direction) on the way to asub-sampling device, and after subsampling, the in-line filter can bebackflushed by the remainder portion during return of the remainderportion in the remainder-return flow path (reverse direction). Moredetails about filtering are described below. In this latter-describedembodiment, the repetitive nature of the aspirating of samples andreturning of remainder portions can be viewed as a repetitive pulsatingof fluid (with repetitive alternating flow in the forward and reversedirections) through the same sampling flow path.

The above description details the methodologies by which the sampledaliquots are subsampled to obtain accurate, reproducible very smallvolume samples as sub-aliquots of the solution, and also to therebyresult in the formation of a remainder portion of the sampled aliquots,and the methodologies by which the remainder portion of the sampledaliquots is returned to the solution. Such methodologies can beadvantageously effected using the system described below in connectionwith the preferred embodiments of the invention.

In particular, such methodologies can be effected using, in a preferredembodiments, using an automated sampling probe for sampling a portion ofthe solution from the container at least twice during the dissolutionperiod, and to obtain first and second aliquots of the solution. Thesampling probe preferably has a distal end positionable in fluidcommunication with the solution in the sample container, and a proximateend that is in fluid communication with a sub-sub-sampling device thatis configured for subsampling a portion of each of the sampled aliquotsof the solution to obtain sub-aliquots of the solution. The automatedsampling probe is preferably a component of an automated liquid handingunit or system comprising the sampling probe together with a robotic armfor translating the sampling probe for positioning, and a pump in fluidcommunication (e.g., selectable or continuous fluid communication) withthe sampling probe. The pump provides a motive force at least forwithdrawing a portion of the liquid sample into the probe to effectsampling (i.e., aspirating a sample). In some embodiments, the same pumpor a different pump can provide a motive force for returning theremainder portion of the subsampled aliquots to the sample container.The pump can be a positive-displacement pump, such as reciprocatingpump. In preferred embodiments, the pump is a syringe pump.

The sub-sampling device is preferably a sampling valve that providesfluid communication between the proximate end of the sampling probe andthe pump. The sampling valve can be a multi-port sampling valve havingat least one sample loop, with the volume of at least one of the sampleloops corresponding to the size of the desired sub-aliquot of thesample. The sampling valve can be advantageously configured so that thefollowing are acheived: in one selectable position, an aliquot of thesolution is aspirated using the sampling probe and at least part of thesampled aliquot is loaded into the sample loop through a sampling flowpath from the proximate end of sampling probe towards the first pump(e.g., in a forward direction); in another selectable position, aremainder portion of the subsampled aliquot is retuned to the containerthrough a remainder-return flow path from the first pump towards theproximate end of the sampling probe (e.g., in a reverse direction), butwhich bypasses the sample loop; and in one selectable position (that canbe the same as the earlier-noted positions or different from one or boththereof), the contents of the sample loop are discharged from thesampling valve as a sub-aliquot of the sampled aliquot (e.g., sent to ananalyzing unit). The volume of the sample loop can be of any volumesuitable for detection, but can generally be as small as less than about100 μl, preferably less than about 50 μl, and in some cases, less thanabout 20 ul, less than about 10 ul, less than about 5 ul, or less thanabout 1 ul.

The operation of the sampling probe, and the operation of thesub-sampling device (e.g., multi-port sampling valve) are preferablycontrolled by a microprocessor under automation software.

Make-Up of Liquid Media (Feature G)

The method can further comprise providing make-up aliquots of a liquidmedia to the solution, with each of the make-up aliquots having a volumeabout the same as the volume of the sub-aliquots that are obtained fromsubsampling of the sampled aliquots withdrawn from the container. Inthis manner, particularly when used in connection with returning theremainder portion of the subsampled aliquots to container, the overallvolume of the dissolving sample in the container can be maintained to besubstantially the same over time. This makes the determination ofconcentration to be more directly comparable between samples, withouthaving to adjust for a change in concentration due to depletion ofoverall volume of the liquid in the dissolution container. Hence, in apreferred protocol, a first make-up aliquot is provided to the containerafter sampling to obtain the first aliquot of the solution, and beforesampling to obtain the second aliquot of the solution. The solution isthen sampled again to obtain the second aliquot, the second aliquot issubsampled to obtain the second sub-aliquot, the remainder portion isoptionally returned, and a second make-up aliquot is provided to thecontainer, preferably before a third or subsequent aliquot is withdrawnfrom the sample container.

The liquid media used as the make-up volume can be the same as thesolvent into which the sample material was dissolved. As noted below,however, the make-up liquid media can be different in chemicalcomposition than the original solvent, such that repetitive addition ofmake-up aliquots can result in a time-varying change in the solvent towhich the sample material is exposed during the dissolution period. Forexample, each make-up aliquot can add an additional amount of an agentthat changes a property of the dissolution solvent environment, such aspH, ionic strength, etc.

The above description details the methodologies by which a make-upvolume, preferably corresponding to the very small volume taken assub-aliquots of the solution, can be added to the container during thedissolution period. Such methodologies can be advantageously effectedusing the system described below in connection with the preferredembodiments of the invention.

In particular, such methodologies can be effected using for example, adispensing probe (e.g.,such as the dispensing probes described above inconnection with initiation of dissolution) to provide the make-up volumedirectly to the container. In an alternative or supplemental embodiment,such make-up volumes can be provided using the automated sampling probe,in combination with the multi-port sampling valve as described above.For example, the sampling valve can be configured in a selectableposition for loading a make-up liquid media into a sample loop, and inanother selectable position, for providing the make-up liquid media tothe container, for example, through a flow-path that is the same as theremainder-return flow path from the sampling valve to the proximate endof the sampling probe, but which is aligned through the sampling valveto a different source reservoir of make-up liquid media and a differentpump. The sample loop for the make-up liquid media can be the same asthe sample loop for subsampling to obtain the sub-aliquot of thesolution. Further details and variations in such systems are describedbelow.

Agitation (Feature H)

As noted, the dissolution profiling methods and systems of the presentinvention will be particularly advantageous in connection withdispersions comprising a solid sample material that is partiallydissolved in a solvent to form a solution, and that is partiallyundissolved and dispersed in the solution. Such dispersions (orsuspensions) can be agitated or mixed. Such agitation or mixing can helpensure sufficient contact between the undissolved solid sample materialsand the solution during the dissolution period, and can provide aphysical averaging of the solutions with respect to the property ofinterest (e.g., concentration). Such agitation or mixing can also helpmaintain the dispersion, preferably as a substantially uniformdispersion. Preferably, the dispersion is sufficiently uniform that itis free of continuous stratification layers that would be of a thicknessthat reflects in a change in a bulk property (e.g. optical turbidity,density) of one layer versus another layer.

The particular type or nature of agitation or mixing is not critical tothe invention. For example, the contents of the container can beagitated or mixed by stirring, shaking, rocking, etc. Orbital shakers,magnetic stirring, shaft-driven stirring, etc. can be employed to effectthe agitation or mixing. In particular, such agitation or mixing can beadvantageously effected using the system described below in connectionwith the preferred embodiments of the invention.

Filtering (Feature I)

As noted above, each of the sampled aliquots of the solution can befiltered during or after sampling to obtain filtered sampled aliquots ofthe solution. Preferably, such filtering is effected during or aftersampling, but prior to subsampling. Filtering is particularlyadvantageous with respect to applications for evalutating thedissolution profile of a sample material in a dispersion, so thatundissolved sample materials can be retained in the solution rather thanincluded in the sampled aliquot. This is particularly significant forevaluations done with relatively small amounts of sample material (e.g.,less than 10 mg as described above), since removing undissolved samplematerials from such dispersions can have affect the dissolution ratebeing determined.

The above described filtration methodologies by which sampled aliquotsare filtered can be advantageously effected using filtering systemsknown in the art. In preferred approaches, the filtering can be effectedusing the system described below in connection with the preferredembodiments of the invention.

In particular, such filtering can be effected using for example, anin-line filter that is integral with or in fluid communication with theproximate end of an automated sampling probe, where as above, thesampling probe has a distal end and a proximate end, the distal end ofthe sampling probe being positionable in fluid communication with thesolution in the sample container for sampling a portion of the solutionto obtain sampled aliquots. Hence, the filter can be in-line in thesampling flowpath between the container and the sub-sampling device. Inpreferred protocols, the remainder portion of the subsampled aliquotsare returned from the sub-sampling device to the solution through thesame flow path as the sampling flowpath, but in the reverse direction,such that the remainder portions of the sub-aliquots backflush thein-line filter while being returned to the solution in the reverse flowdirection. Such “pulsating” action can be rapidly effected using theapparatus and method disclosed below in connection with the preferredembodiment.

Sampling Frequency/Sampling Plan (Features J, K)

Subsequent aliquots of the solution are preferably sampled with arelatively fast sampling frequency, where the sampling frequency isdefined by the difference in time between sampling of successivealiquots (e.g., the difference between the first time and the latersecond time at which the first aliquot and the second aliquot aresampled, respectively). In preferred protocols, the sampling frequencyis not more than about 2 minutes, and more preferably not more thanabout 1 minute. For many applications, including for example forinvestigations into drug candidates or drug compositions, the samplingfrequency for at least two successive samples, and preferably for atleast three successive samples, can be even faster, including forexample not more than about 1 minute, preferably not more than about 40seconds, or in some cases not more than about 30 seconds, not more thanabout 20 seconds or not more than about 10 seconds.

It will be appreciated by a person of skill in the art that the samplingfrequency can depend on the nature and throughput of the analyticalprotocol and analytical unit being used (e.g., for determining theconcentration of the sample material in the solution). Generally forapplications involving analytical protocols/units involving separationof the subsampled subaliquot into one or more components (e.g., via oneor more HPLC columns), the sampling frequency will preferably range fromabout 30 seconds to about 2 minutes, and preferably from about 30seconds to about 1 minute. For applications that are not involvingseparation of the subsampled subaliquot into one or more components(e.g., via flow-injection analysis), the sampling frequency can behigher, including for example, ranging from about 10 seconds to about 2minutes, and preferably from about 10 seconds to about 1 minute or fromabout 10 seconds to about 40 seconds or from about 10 seconds to about30 seconds.

In particularly preferred embodiments, the protocols of the inventioncan include a sampling plan for efficient sampling, particularly forefficiently sampling multiple members of an array of sample materials.In one embodiment, for example, a sampling plan includes for eachsample, sampling the solution at least six times, and preferably over atleast two distinct sampling intervals. A first sampling intervalcomprises at least three sampled aliquots at three successive times andis, as described above (in connection with Feature D), preferablyproximate in time with the initiation of dissolution, so that an initialpart of a dissolution profile can be obtained for a sample material. Alater-in-time second sampling interval also preferably comprises atleast three sampled aliquots at three successive times. The samplingfrequency within the first sampling interval, and within the secondsampling interval is preferably less than about 1 minute, and evenfaster as described above (in connection with Feature J). Thenon-sampling interval that defines the time period between the end ofthe first sampling interval and the beginning of the second samplinginterval is preferably at least not less than two times the greatestsampling frequency used in connection with any adjoining samplinginterval, and more preferably three or four or five or six or seven oreight or nine or tent times such sampling frequency. In someapplications, including for example for evaluating dissolution of drugcandidates or drug compositions, the non-sampling interval can be atleast sufficient to allow for the asymptotic maximum solubility to beapproached for each solution system being evaluated (e.g., for thatamount of solute in that amount of solvent at that temperature(s)). Thissampling plan is advantageous in that it obtains data from an early partof the dissolution, substantially proximate in time with the initiationof dissolution, and then obtains data from a much later part of thedissolution, where the time-rate-of-change of the concentration of thesample material is much lower than at the early part of the dissolution,and therefore, with as few as six sampled aliquots, can establish asubstantial portion of the dissolution profile. The advantage of suchsampling efficiency is particularly noted with regard to evaluatingsmall amounts of sample materials.

A further advantage of such a sampling plan is that it allows forinterleaved sampling protocols that can be advantageously applied inconnection with the evaluation of multiple members of an array of samplematerials. For example, in an array comprising four or more multiplesample materials, each of the sample materials could be sampled withrespect to the first sampling interval (as described above), with thenthe second sampling interval (as described above) being initiated afterthe first sampling interval is completed for each of the four or moresample materials.

In another embodiment of a sampling plan for efficient sampling,particularly for efficiently sampling multiple members of an array ofsample materials, one may be less interested in sampling immediatelyafter initiation, but rather, sampling more frequently after aninduction period (e.g., allowing time-delayed disintegrants to takeeffect). In such a case, there may again be only a single samplinginterval (temporally sequenced near the inflection point of thesolubilization curve or dissolution profile), or two or more samplingintervals or three or more sampling intervals. Such a plan can include,for example, a first sampling interval comprising at least one sampledaliquot to establish a baseline point, preferably proximate in time withthe initiation of dissolution, and a later-in-time second samplinginterval preferably comprising at least two, more preferably at leastthree sampled aliquots at three successive times to establish theinflection point or other point at which the maximum time-rate-in-changeof determined concentration exists. The jplan may further include alater-in-time third sampling interval preferably comprising at least onesampled aliquot to establish an asymptotic maximum value (e.g.solubility) to be approached for each solution system being evaluated(e.g., for that amount of solute in that amount of solvent at thattemperature(s)).

Dissolution Environment

The dissolution environment—including for example, the composition ofthe solution in which dissolution occurs during the dissolution period,and/or the temperature or other properties during the dissolutionperiod, and/or the temperature and/or humidity and/or other environmentsto which a sample material is exposed prior to the dissolutionperiod—can be controlled, including as a parameter of the evaluation.Temperature is a particularly preferred parameter to control during adissolution period, since solubility and solubilization rate can vary asa function of temperature. For example, the protocols (including themethods and systems) of the invention can be adapted to control thetemperature of the solution at a desired temperature or within a desiredrange of temperatures, typically defined by upper and lower setpoints ofa temperature controller. In some applications, for instance, one cancontrol the temperature to be at or near or including ambient or roomtemperature (e.g., about 25 deg. C.) or at or near or including normalbody temperature (e.g., about 37 deg. C.). If an array comprisingmultiple sample materials as members are being evaluated, for examplewhile residing on a common substrate, the array (e.g., the substrate)can be controlled individually, as subgroups or groups, or as an arrayas a whole. For example, the array can be enclosed in an environmentalcontrolled chamber so that the atmospheric environment (e.g.,temperature, humidity, and the like) of the members of the array can becontrolled. Environmental control, including temperature and/or humiditycontrolled chambers, is described in Ser. No. 60/451,463 entitled “NovelMethods and Apparatus for Evaluating the Effects of Various Conditionson Drug Compositions Over Time” filed Mar. 1, 2003 by Carlson et al.

Dynamic Dissolution Environment (Feature L)

The dissolution environment—such as especially the composition of thesolution in which dissolution occurs during the dissolution period,and/or the temperature or other properties during the dissolutionperiod—can be maintained to be substantially the same, or can be varied,and preferably controllably varied over time during the dissolutionperiod. Advantageously, such capabilities can be used to obtain adissolution profile that is representative of dissolution in a dynamicenvironment. In one non-limiting example, it may be desirable to mimicthe environment along various regions of the gastrointestinal tract. Forexample, the pH of dissolution solution intended to mimic thegastrointestinal tract may be controllably varied from about neutral orslightly basic (e.g., representing the mouth at pH of about 7 orslightly higher), to more acidic (e.g., representing the stomach at pHranging from about 2 to 4), to more basic (e.g., representing theintestines at pH ranging from about 7 to 9). Other properties that canbe varied, and preferably controllably varied include temperature, ionicstrength, ratio of solvent to co-solvent, solubilizer concentration,disintegrant concentration, surfactant concentration, wetting agents,etc.

Such variation can be effected by adding a liquid-media as a make-upagent, as described above, where the liquid-media is effective forchanging one or more of said solution properties over time.Alternatively or supplementally, solid or liquid agents or co-solventscan be added directly to the solutions, for example using a dispensingprobe as described above.

Analysis

The sampled aliquots of the solution (or in preferred embodiments, thesubsampled sub-aliquots of the solution), are analyzed to determine theconcentration of the sample material in the aliquots. The determinedconcentration of each of the successively-sampled aliquots, whenconsidered in combination with the corresponding times at which thesolution was sampled to obtain the aliquots, represents a dissolutionprofile for the sample material being dissolved.

Where the surface area of the sample material is known or determined,the methods can further comprising determining an intrinsic dissolutionrate for the sample material for one or more times during thedissolution period. Alternatively, where the surface area of the samplematerial is not known, or where a screening application (comparativeevaluation between two or more sample materials) is desired, such as incombinatorial applications involving an array comprising multiple samplematerials, the method can further comprise comparing the relativedissolution rates for the sample materials being evaluated for one ormore times during the dissolution period.

The particular manner of analysis is not narrowly critical to theinvention. Analysis can be effected, for example, using techniques knownin the art. Generally, the analysis methods can be classified as thoseinvolving separation of one or more components of a sampled aliquot orsub-aliquot from other components thereof, and those that do not involvesuch separation. The analysis methods can also be classified as thoseadaptable for use in connection with a to-be-analyzed aliquot in a flowmode (e.g., analyzers having flow detectors of capable of analyzingsamples coming from a flow system).

Analysis can be effected using an analytical system, such as theanalytical system described below in connection with the preferredembodiments of the invention. In particular, such analysis can beeffected using for example, an analytical unit comprising a detector,and in some cases a flow detector, each of which are in fluidcommunication with the sub-sampling device (e.g., sampling valve). Thedetector is adapted for detecting a property of the one or moreseparated components. The detector can be selected from one or more of alight-scattering detector (e.g., especially a static light-scatteringdetector, a dynamic light-scattering detector and/or a evaporativelight-scattering detector (“ELSD”), a refractive index detector, afluorescence detector, an infrared detector, and a spectroscopicdetector (e.g., especially a ultra-violet/visible (“UV/VIS”) absorbancedetector. The analytical unit can further comprise a separation devicein fluid communication with the sub-sampling device. The separationdevice can be adapted for separating one or more components of thesubsampled sub-aliquots discharged from the sampling valve from othercomponents thereof. Specifically, the separation device can be a liquidchromatography column.

Preferred analytical units can include a separation unit. The separationunit of the analytical unit be any device that can effectively separatecomponents of the sub-sampled aliquot of the solution, including forexample, separation units such as high performance liquid chromatography(HPLC), liquid chromatography (LC), gel permeation chromatography (GPC),gas chromatorgraphy (GC), capillary electrophoresis (CE), capillaryelectrochromatography (CEC), and the like. Thin-layer chromatography(TLC) can also be employed. Especially preferred analytical unitsinclude a liquid chromatography unit or a flow-injection analysis unit.The flow-injection analysis unit can be a continuous flow, stop-flow orvariable flow system.

Combinatorial Research with High-Throughput Dissolution (Feature M)

The aforementioned protocols (including specific methods and/or devices)can be applied to a high-throughput method and system for evaluating alibrary or an array of sample materials. An array is an association oftwo or more, preferably four or more, most preferably eight or moremembers, such as sample materials. The array can comprise multiplemembers of sample materials in containers formed on or supported by acommon substrate.

The aforementioned protocols can be effected in connection with each ofthe plurality of members of the array, generally either sequentially intime (i.e., serially), or simultaneously in time (i.e., in parallel).Also, it is possible that some steps of the methods are effectedserially (e.g., sampling of aliquots), while other steps of the methodsare effected in parallel (e.g., analysis), in each case as comparedbetween different members of the array. The members of the array can bevaried from each other in a number of ways, including for examplediffering with respect to chemical composition, differing with respectto crystalline structure (polymorphs), etc. In some embodiments, thematerial samples that are members of an array will be chemically andphysically substantially the same (e.g., have the same chemicalcomposition and same crystalline structure), and the method willcomprise creating a different dissolution environment (e.g., differentsolvents, different temperatures, different ratios of cosolvents,different concentrations of additives) for each of the members of thearray.

The array of library members for dissolution screening can beadvantageously prepared using high-throughput formulation methods andsystems. These can be accomplished for example, by using a formulationstation such as that described by art disclosed methods and apparatusincluding, without limitations, the methods and apparatus described indetail in the PCT Publication No. WO 03/014732, and in U.S. Ser. Nos.10/156,222, 10/156,245, 10/156,329, now abandoned, and 10/156,295 and inthe commonly owned and co-pending PCT Publication Nos. WO 00/23921; WO02/31477 A2, and WO 02/14391 A2, which are hereby incorporated byreference in their entirety. See also PCT Publication No. WO 99/52962and U.S. Ser. No. 09/640,094 entitied “Procedure And Device To DevelopNanodispersants” filed Aug. 17, 2000 by Sebrof et al., which is herebyincorporated by reference.

For example, formulation by wet and/or dry milling can be used. Suchformulation is comprised of dispensing with an art disclosed liquidhandling robot all components such as drug candidate, excipients,stablilizers, surfactants, and the like into an array of vials. If asolvent is used to dispense the components, it can be stripped offeither by vacuum or by blowing dry nitrogen directly on the array.Milling media is then added to the array by means of solids dispensing.The milling media can be any art-disclosed milling media such as glassbeads of various sizes (e.g., 0.5 mm to 6 mm), stainless steel beads ofvarious sizes (e.g., 0.5 mm to 6 mm), polymer beads (e.g., PS-DVB beads)of various sizes (e.g., 50 um-1 mm), and combination thereof. Wetmilling is accomplished by adding an art-disclosed suitable aqueousmedia along with appropriate art-disclosed wetting agents. Milling ispreferably done in parallel with the use of a 5 g Harbil mixer but othersuitable art-disclosed mixers may also be used. Various degrees ofmixing can be achieved by using various combinations of mixing media andvarious mixing times. After the mixing is completed, the formulation isseparated from the milling media preferably by centrifugation and/orfiltration.

The formulation process can be controlled by computer to automaticallymix specific quantities of material (e.g., drug candidates, solvents,stabilizers, etc.) to form a material composition. The materials may bemixed in accordance with library designs produced by computer—preferablywith library design software such as Library Studio® (SymyxTechnologies, Inc., Santa Clara Calif.). Automatic control of theformulation station 1130 enables the present invention to prepareseveral libraries or members. Moreover, automated control reducespossible errors that can be caused by manual control. In addition,automated control may enable the present invention to prepare relativelysmall sample sizes (e.g., ranging between nanoliter to millilitersizes). This advantageously provides high throughput preparation andscreening of the material compositions.

After the array of multiple sample materials are prepared, they may bescreened by the dissolution methods disclosed herein. Specfically, ahigh throughput drug solubilization characterization or screening methodand system is provided to identify, select, synthesize, and the form ofa drug candidate having desirable dissolution properties. Thissolubilization screening method generally comprises: (1) providing alibrary comprising a plurality of members (i.e., sample materials),wherein each said library member comprises a drug candidate; (2)determining a dissolution profile for each of the plurality of librarymembers for solubilization characteristics using any of the methodsdetailed in this application for determining a dissolution profile; and(3) comparing the dissolution for each of the plurality of librarymembers. The solubilization screening system generally comprises: (1) aplurality of sample containers, preferably four or more samplecontainers, each for containing one member of the library or array forevaluation—either separately or structurally integrated (e.g, beingformed in or supported on a common substrate); (2) one or more automatedsampling probe; (3) an analytical unit that is either aserial/single-channel unit—adapted to analyze one sample material at atime, and a plurality of sample materials sequentially, or aparallel/multi-channel unit—adapted to analyze two or more samplessimultaneously. Preferably such system also includes (4) one or moreautomated dispensing probes. In one parallel embodiment, thesolubilization screening system comprises two or more sets ofstructurally integrated or structurally independent automated probes,each set comprising an automated sampling probe and an automateddispensing probe (e.g., each set being structurally integrated through acommon probe head).

Such high-throughput solubilization screening can be applied alone or incombination with various other screening methods for evaluating otherproperties of interest, such as stability, compatability, solubility,crystallinity, particle size, etc. Taken in combination as part of alarger workflow, such screens can systematically enhance the efficiencyof the process of drug development.

In preferred embodiments, all of the above-described steps of thehigh-throughput solubilization screening methods are automated andcontrolled by a computer. The formulation of libraries can also beautomated and controlled by a computer. Preferred automation software isImpressionist® (Symyx Technologies, Inc., Santa Clara Calif.). Userinterfaces can enable users to input commands to computer via an inputdevice—including any suitable device such as, for example, aconventional keyboard, a wireless keyboard, a mouse, a touch pad, atrackball, a voice activated console, or any combination of suchdevices. Input device enables a user to enter commands to perform drugselection, library building, screening, etc. If desired, input devicemay also enable a user to control the various workstations (e.g., forformulation or for solubilization screening). A user may also monitorprocesses operating on the systems on a display device, such as acomputer monitor, a television, a flat panel display, a liquid crystaldisplay, a cathode-ray tube (CRT), or any other suitable display device.Communication paths can be provided and configured to enable datatransfer among the computer, the formulation workstation, thesolubilization screening workstation, and user interfaces.

Preferred Embodiment for High-Throughput Dissolution Screening

Referring now to FIG. 1, the system 100 of the present invention isgenerally comprised of an automated dispensing unit 14, an automatedsampling unit 20 and an analytical unit 34.

The automated dispensing unit 14 uses an automated dispensing probe 15to dispense a first liquid 16 (e.g., a solvent) into a container formedas a well 11 of a substrate 10, where an amount of at least one samplematerial 12 is located. As shown, the automated dispenser is a anautomated liquid dispenser that also comprises a pump 17 as a motiveforce for moving the liquid, and a probe head 19 configured with arobotic arm (not shown) and/or translation station (not shown) forpositioning the probe head 19, and thereby a distal end of thedispensing probe 15 into or over the container, shown here as well 11.Dispensing of the first liquid (e.g. solvent) 16 into a well 11 thatalready contains the sample material 12 initates dissolution of thesample material into the solvent, to form the solution 18. Preferredliquid handling robots that can be used as the dispensing unit 14include those sold by Tecan Systems (formerly Cavro ScientificInstruments) (San Jose, Calif.).

The automated sampling unit 20 shown in FIG. 1 comprises an automatedsampling probe 21 for sampling a portion of the solution successivelyduring the dissolution period. The sampling probe has a distal end 21 apositionable in fluid communication with the solution in the samplecontainer, and a proximate end 21 b, and is generally shown as beingsupported by the probe head 19. The proximate end 21 b of the samplingprobe 21 is in fluid communication with a sub-sampling device 24, thesub-sampling device (e.g., sampling valve) is configured for subsamplinga portion of each of sampled aliquots of the solution to obtainsub-aliquots thereof. The sampling unit 20 also includes an in-linefilter (i.e., separation medium) 32 positioned in fluid communicationwith and in the flow between the sampling probe 21 and the sub-samplingdevice 24. Positioning of the sampling probe 21 is effected using theprobe head 19 in combination with a robotic arm (not shown) or/or atranslation station (not shown). A pump 22 (e.g., a syringe pump) isshown in selectable fluid communication with the proximate end 21 b ofthe sampling probe 21 via sub-sampling device 24, for providing a motiveforce for withdrawing a portion of the solution into the probe 21 toeffect sampling through a sampling flow path (in a forward direction),and in preferred embodiments, for returning remainder portions of thesubsampled aliquots to the containers through a remainder-returnflowpath (in a reverse direction) and/or for providing a make-up aliquotto the sample container through an make-up flow path (in the reversedirection). Although the pump 28 is shown as part of the analytical unit34, this pump 28 can instead, or also be part of the sampling unit 20,in that context, can be considered as a second pump 28, for providing amake-up aliquot (i.e., replacement volume) of a second liquid media 30into the solution 18 located in the container shown as well 11 on thesubstrate 10. The multi-functionality of pump 28 can depend, as is wellknown in the art, on the particular configuration of the sub-samplingdevice (e.g., sampling valve), some of which are described furtherbelow.

The analytical unit 34, as shown, comprises a detection device 27 thatcan include a detector 26, or multiple such detectors 26, that one ormore of which can be a flow detector, but can also be aspatially-sensitive detector that operates in a non-flow mode (e.g. aregion of pixels of a CCD camera). The analytical unit 34 can alsocomprise a mobile phase source reservoir 30 for containing a liquidmedia as a mobile phase (e.g., of a liquid chromatography system or of aflow-injection analysis system), and a pump 28 for providing a motiveforce for effecting flow of the mobile phase through the sub-samplingdevice 24 (e.g., sampling valve), to the detector 26.

Referring to FIG. 2, a preferred embodiment 400 of the dispensing unit14 can comprise an injection probe or dispensing tip 402 mounted on arobot arm 404, a microprocessor 406 for controlling three-dimensionalmotion of the tip 402 between various spatial addresses, and a pump 408for withdrawing the first liquid 16 into the tip 402 and dispensing thefirst liquid 16 onto the substrate 10 where the at least one sample 12is located. The microprocessor 406 is preferably user-programmable toaccommodate varying arrangements of the at least one sample 12 (e.g.,square arrays with “n-rows” by “n-columns”, rectangular arrays with“n-rows” by “m-columns”, round arrays, triangular arrays with “r-” by“r-” by “r-” equilateral sides, triangular arrays with “r-base” by “s-”by “s-” isosceles sides, etc., where n, m, r, and s are integers). Thetip 402 has a surface defining a cavity and a port for fluidcommunication between the cavity and the substrate 10. The tip 402 alsocomprises a port for fluid communication between a receptacle 10 for thefirst liquid 16 and line (not shown) and the cavity.

The dispensing unit 14 may further comprise a temperature-controlelement (not shown) in thermal communication with the tip 402 formaintaining the liquid 16 residing in the tip 402 at a predeterminedtemperature or within a predetermined range of temperatures. Thetemperature-control element can be, in the general case, a heatingelement or a cooling element (for low-temperature characterizations).The particular design of the heating element or cooling element is notcritical. For example, the heating element can be a resistive-heatingelement such as a resistive wire in adjacent proximity to the samplecavity of the tip 402. The heating element can alternatively be afluid-type heat-exchanger heating element having a fluid-containingtubular coil around the tip 402. In any case, the temperature-controlledtip 402 can have a body encasing the heating element, and preferably athermocouple for temperature monitoring and control. In anotheralternative embodiment, the heating element can be the body of the tip402 itself, where the body comprises a large thermal mass, preferablysurrounded by an insulator. The large-thermal-mass body can be heated(or in the general case, cooled) by periodically allowing the body tothermally equilibrate with a hot environment such as a surface or fluidvia conduction, convection or thermal radiation (or generally, with ancold environment). Advantageously, such a heated tip 402 can maintainthe first liquid 16 at the required temperature while it resides in thecavity of the tip 402. As such, unlike conventional high-temperaturecharacterization systems, the tip 402, as well as associated robotic arm404, can be located external to (outside of) a heated environment (e.g.,oven). Another example of a preferred dispensing unit 14 is furtherdescribed in detail in commonly owned U.S. Pat. No. 6,175,409 B1, whichis incorporated herein by reference in its entirety.

It is also preferred that a controlled agitation assembly 36 is includedin the dispensing unit 14 to agitate the solution 18 during dispersion.The controlled agitation assembly can be any art disclosed device thatprovides agitation of the solution 18 such as magnetic stirrer, orbitalshaker, parallel ball milling, rocker, sonicating probe, homogenizer,and the like. For example and referring to FIG. 3, a controlledagitation assembly 502 is included in the dispensing unit 500 having aresonant sealed enclosure 504 integrated into the dispensing unit's 14base 506, and a voice coil motor 508 comprising of an audio subwoofer510 (e.g., 1,500 W dual-coil audio subwoofer) and audio amplifiers 512(e.g., two power amplifiers) providing a controlled variable frequency(e.g., 20 to 500 Hz) and amplitude (0–20 mm). In a preferred embodiment,the controlled agitation assembly is comprised of orbital shakersoperating at a variable frequency, typically around 100 Hz, to providevertical agitation of the solution 18.

Referring to FIG. 4, preferably also in connection with FIG. 1, theanalytical unit 34 is used to determine the concentration of aliquots orsubaliquots 25 drawn successively over time from a solution having avarying concentration of the sample material 12. The analytical unit 34can be any art-disclosed device that is effective for determiningconcentration, such as shown in FIG. 4 as concentration detector602—including such as an ultra-violet (UV) visual inspector, arefractive index detector, an infrared detector, an evaporative lightscattering detector, a fluorescence detector, and the like. Theconcentration detector 602 is preferably a highly sensitive detector,being adapted for and with capability to detect concentration levelseven in the range of about 0.0001 mg/ml to about 10 mg/ml.

Referring further to FIG. 4, in a preferred embodiment, the analyticalunit 34 may further include a microprocessor 604 that records andtranslates the data obtained from concentration detector 602 intographic and/or tabular numerical form (e.g., dissolution curve ordissolution profile) and can provide other information regardingsolution 18. The same microprocessor 604 can also be used in connectionwith control of the automated dispensing unit 14 and/or the automatedsampling unit 20. In a preferred embodiment, a separation device 606 isalso included as a component of the analytical unit 34 and can belocated upstream from the concentration detector 602. The separationdevice 606 (e.g., chromatography column) is used to separate and isolateone or more components of (e.g., various compounds contained in) thesampled aliquot or subsampled sub-aliquot 25 of solution 18, fordetection by the concentration detector 602.

Since the separation process by the separation device 606 may in somecases require a longer time period than the time required for thesampling process by the sampling unit 20, it may be desirable to haveparallel separation devices, with a single common injection valve. See,for example, U.S. Pat. No. 6,296,771, which is hereby incorporated byreference in connection with the apparatus and techniques disclosedtherein. Alternatively, rapid-serial high-throughput techniques can beemployed. See, for example, in connection with characterization ofnon-biological polymers, U.S. Pat. Nos. 6,406,632 and 6,461,515.

The sub-sampling device 24 is preferably a sampling valve, such as amultiport switching valve having at least one sample loop. The samplingvalve or switching valve 24 can be constructed out of any suitablematerial such as metals, (e.g., stainless steel, aluminum, and thelike), plastic, and ceramics. Materials such as aluminum or an aluminumalloy may be preferred because they have desirable thermal andstructural properties.

One configuration for the sampling valve 24 is shown in FIG. 5A.Briefly, as shown, sampling valve 24 is a multi-port sampling valvecomprising a sample loop 702 and at least six ports (shown as beingnumbered 1–6), including a first port 1 in fluid communication with theproximate end (21 b, FIG. 1) of the sampling probe (21, FIG. 1), and asecond port 2 in fluid communication with a first pump 22, a third port3 in fluid communication with a first end 702 a of the sample loop 702,a forth port 4 in fluid communication with a second end 702 b of thesample loop 702, a fifth port 5 in fluid communication with ananalytical unit 34, and a sixth port 6 in fluid communication with asecond pump 28.

The sampling valve 24 is configured so that in a first selectableposition (shown as “Position A”), the first and third ports are in fluidcommunication with each other, and the fourth and second ports are influid communication with each other, for loading at least part of thesampled aliquot into the sample loop 702 through a sampling flow pathfrom the solution 18 through the sampling probe (21, FIG. 1) towards thefirst pump 22. The sampling valve 24 is also configured so that in asecond selectable position (shown as “Position B”), the sixth and fourthports are in fluid communication with each other, and the third andfifth ports are in fluid communication with each other, for dischargingthe contents the sample loop 702 as a sub-aliquot of the sampled aliquotthrough a detection flow path from the second pump 28 to the detector34. The sampling valve 24 is further configured so that in the secondselectable position (shown as “Position B”), the second and first portsare in fluid communication with each other, for returning a remainderportion of the sampled aliquot not loaded into the sample loop 702 tothe solution 18 in the container (e.g., well 11, FIG. 1) through aremainder-return flow path from the direction of the first pump 22through the sampling probe (21, FIG. 1) to the solution 18.

Referring now to FIG. 5B, another configuration is shown for thesampling valve. The sampling valve 24 as shown therein comprises firstand second sample loops 702, 704 respectively, that can be used tosubsample an aliquot to form a sub-aliquot for (optional separation) andfor analysis by the analytical unit 34. While the sampling valve 24 isin one selectable position (e.g., position “A”), a first pump 22aspirates a portion of the solution 18 contained in a well 11 formed inthe substrate 10. The aspirated aliquot is drawn through filter 32 (FIG.1), and a portion of the filtered sampled aliquot is then loaded intothe sample loop 702. When the valve 24 is switched to a secondselectable position (e.g., position “B”), the sub-aliquot loaded in thesample loop 702 is then discharged by the second pump 28 as sub-aliquot25 to the analytical unit 34, the discharge path as shown in FIG. 5Bincluding the detection flow path from the first sample loop 702,through the second sample loop 704 to the analytical unit 34. Meanwhile,with the sampling valve 24 in this position B, the first pump 22 returnsthe remainder portion of the filtered sampled aliquot to solution 18 inthe container (well 11) through the sampling valve 24, but bypassing thesample loop 702. This process is repeated for each of the aliquots thatis desired to be analyzed by the analytical unit 24. Note that sampleloop 704 is functionally extraneous in this embodiment. Also, if amake-up aliquot is desired, it is not provided through the samplingvalve 24 in this embodiment, but rather could be provided directly froman automated dispensing unit 14 (FIG. 1).

FIG. 6 provides another exemplary embodiment of a sampling valve 24,with multiple ports and two sampling valve. This sampling valve 24affords the additional functionality of allowing for a make-up volume tobe directly determined using the same sample loop as is used forsub-sampling to obtain sub-aliquot 25. While the valve 24 is in oneselectable position (e.g., position “A”), the first pump 22 aspiratesthe solution 18 contained in well 11 formed in the substrate 10 throughthe valve 24, allowing a portion of the sampled aliquot to be loadedinto the first sample loop 802. When the valve means is switched to asecond selectable position (e.g., position “B”), a second pump 806 loadsa make-up aliquot into the first sample loop 802, which in turn advancesthe sub-sampled sub-aliquot to a second sample loop 804. Meanwhile, thefirst pump 22 returns the remainder portion of the solution 18 backthrough the valve 24 into the sample container 11. Thereafter, thesampling valve 24 is switched back to the original position (e.g.,position “A”) at which point the pump 28 (which can be incorporated intoor separate from the analytical unit 24) discharges the sub-aliquot 25from the second sample loop 804 to the analytical unit 34. At the sametime, the first pump 22 can provide the make-up aliquot from the firstsample loop 802 back to the solution 18. This process is repeated foreach of the sub-aliquots 25 that is desired to be evaluated by theanalytical unit 24.

Although the above-described invention has been described for drugcandidate compounds as the sample material, this invention may bepracticed with any compound of interest as the sample material. Thus,the methods and systems described herein may be used for any element,compound or composition for which a determination of dissolution profileis desired.

The following examples provide illustrative examples on how the presentinvention can be used to perform the processes described above. Theseexamples are for the purpose of illustration only and are not to beconstrued as limiting the scope of the invention in any way.

EXAMPLE 1 High Throughput Dissolution Screening of Aspirin

A 10 mg of powdered Aspirin standard from Aldrich was weighted into a 20ml scintillation vial and put on a shaker at a robot platform. Theshaker was then turned on and an automated dispensing unit with a 10 mlsyringe dispensed 10 ml of deionized water into the 20 ml scintillationvial containing the 10 mg of Aspirin thereby initiating dissolution ofthe 10 mg of Aspirin into the 10 ml of deionized water to form, overtime, a solution having an increasing concentration of aspirin.

Thereafter, an automated sampling unit aspirated approximately 300 uL ofthe solution (the sample itself never enters the syringe) through anin-line filter allowing a sampled aliquot of the filtered solution toenter a switching valve and fill a 20 uL sample loop connected to theports of the switching valve. Once the 20 uL sample loop was filled witha portion of the aliquot, the switching valve was switched to a secondposition by automation, causing the sub-aliquot contained in the 20 uLsample loop to be discharged into an analytical unit. The analyticalunit comprised a syringe pump and an UV detector, for determiningconcentration. At this point, the automated sampling unit returned theremainder portion of the filtered aliquot back through the filter andback into the solution contained in the scintillation vial. Suchbackflush thereby allowed the filter to be cleared and undissolvedparticles to be returned back into the container. Thereafter, theswitching valve was switched back to its original position and thisentire process was repeated with a 37 second sampling frequency betweensuccessive samples for 30 minutes total, yielding the dissolutionprofile shown in FIG. 7. FIG. 7 shows that the typical aliquot toaliquot variation in peak areas is approximately +/−5% and that themaximum dissolution of Aspirin was reached in less than 10 minutes.

EXAMPLE 2 High Throughput Enhanced Drug Solubilization of an ActiveCompound

A library of sample formulations of a poorly water-soluble activecompound was prepared and screened using the methods and system of theinvention. The library design for the experiment is shown in FIG. 8.Three concentrations of the active compound, four ratios of the activecompound to stabilizer, and two stabilizers that differ in molecularweight were used as compositional variables, leading to a 24 welllibrary. The volumes for the design were calculated by Library Studio®and saved to the computer database.

The formulation process was carried out by the formulation stationcomprising of a liquid handling robot equipped with Impressionist®software. (Symyx Technologies, Santa Clara, Calif.). The library designwas read by the Impressionist® software, which then controlled theliquid handling robot to add the stabilizer to the appropriate wells bydispensing a concentrated aqueous solution of each of the stabilizers.The liquid handling robot subsequently topped off all wells to 800 uL.Agitation was turned on, and the liquid handling robot then added theappropriate amount of the volatile organic solution of the activecompound to each well. The library of formulations was stirred for 4hours to allow the volatile organic solvent to evaporate.

Thereafter, each liquid formulation in the library was imaged by thevisual inspection station. The captured images of the library are shownin FIG. 9. Formulations in Row A where there was no stabilizer all grewlarge crystals which settled to the bottom of the vials. As the amountof stabilizer is increased and as the molecular weight of the stabilizeris increased, formulations appeared homogeneous. Wells D3, D4, D5, andD6 were looked the most homogeneous by visual inspection.

Based upon the data obtained from the visual inspection screening, theliquid handling robot of the formulation station was then used todaughter the selected formulations of the library for additionalscreenings. For each selected formulation, the liquid handling robotdaughtered 400 uL to an array of vials for particle size screening, asample to an array of 8 mL vials for dissolution screening, and 100 uLto an assembly holding a substrate for crystallinity screening. Thesubstrate having samples thereon can be used for a variety of screening,including for example, birefringence, Raman, x-ray diffraction, ormelting point, without handling of the individual sample materials.Representative crystallinity data is depicted in FIG. 11. Detaileddescription of the preferred substrate is provided in the PCT/US02/16962, which is hereby now incorporated by reference. The volume ofthe dissolution sample was calculated by Library Studio® based on theconcentration of the active compound so that 0.1 mg of the activecompound was transferred into the dissolution sample.

The 400 uL vials of selected formulations were transported to theparticle size station comprising of a commercial multiangle lightscattering instrument that has been implemented with a roboticautosampler. These vials were screened for particle size. Volume averageparticle size distributions are plotted in FIG. 10. Each graph shows thedistribution from 0.1 um to 100 um on a logarithmic scale. B4 and C2showed bimodal distributions of particle size. The smallest particlesizes were seen in well D4.

After freeze-drying and lyophylization, the array of 8 mL vials holdingaliquots of selected formulations was transported to the solubilitystation for solubility screening using the high throughput solubilityscreening system and methods of the present invention described above.Dissolution profiles and dissolution times of the selected formulationsare shown in FIG. 12.

As the data described above revealed, where there was no stabilizer, nostable formulations were formed. The data showed that as the relativeamount of stabilizer was increased, the average particle size of theformulation decreased, the crystallinity increased, and the dissolutionrate decreased. This workflow also showed that the fastest dissolutionrate correlated with the smallest particle size and in this case thesample formulation with the highest crystallinity. In addition, wells B4and C3 showed bimodal particle size distributions that correlated withdissolution profiles that showed two different dissolution rates in eachsample formulation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for determining a dissolution profile for a sample material,the method comprising dissolving at least a portion of the samplematerial in a solvent over a period of time to form a solution, theperiod of time defining a dissolution period, the solution having aconcentration of the sample material that varies during the dissolutionperiod, sampling a portion of the solution at least twice during thedissolution period, the solution being sampled at a first time withinthe dissolution period to obtain a first aliquot of the solution, and ata later second time within the dissolution period to obtain a secondaliquot of the solution, subsampling a portion of the first aliqout ofthe solution to obtain a first sub-aliquot of the solution resulting ina remainder portion of the first aliquot, determining the concentrationof the sample material in the first sub-aliquot of the solution,returning the remainder portion of the first aliquot to the solution,subsampling a portion of the second aliqout of the solution to obtain asecond sub-aliquot of the solution resulting in a remainder portion ofthe second aliquot, determining the concentration of the sample materialin the second sub-aliquot of the solution, and returning the remainderportion of the second aliquot to the solution.
 2. The method of claim 1further comprising providing a first make-up aliquot of a liquid mediato the solution, the first make-up aliquot having a volume about thesame as the volume of the first sub-aliquot, the first make-up aliquotbeing provided after sampling to obtain the first aliquot of thesolution, and before sampling to obtain the second aliquot of thesolution, and providing a second make-up aliquot of a liquid media tothe solution, the second make-up aliquot having a volume about the sameas the volume of the second sub-aliquot, the second make-up aliquotbeing provided after sampling to obtain the second aliquot of thesolution.
 3. The method of claim 2 wherein the liquid media for each ofthe first make-up aliquot and the second make-up aliquot is a solventthat is the same as the solvent in which at least a portion of thesample material is initially dissolved.
 4. The method of claim 2 whereinthe liquid media for each of the first make-up aliquot and the secondmake-up aliquot is a cosolvent that has a chemical composition that isdifferent from the solvent in which at least a portion of the samplematerial is initially dissolved, whereby the solution has a solventcomposition that varies over time during the dissolution period.
 5. Themethod of claim 2 wherein the liquid media for each of the first make-upaliquot and the second make-up aliquot is pH-adjusting agent, wherebythe solution has a pH that varies over time during the dissolutionperiod.
 6. The method of claim 1 wherein the solution is sampled atleast three times during the dissolution period, the method furthercomprising sampling a portion of the solution at a third time within thedissolution period to obtain a third aliquot of the solution,subsampling a portion of the third aliquot of the solution to obtain athird sub-aliquot of the solution, and determining the concentration ofthe sample material in the third sub-aliquot of the solution, whereinthe third time is after the second time.
 7. The method of claim 6wherein the sampling frequencies defined by the difference in timebetween the first time and the second time, and by the difference intime between the second time and the third time, are each not more thanabout 1 minute.
 8. The method of claim 6 further comprising combiningthe sample material with the solvent at an initiation time, t_(o), toinitiate dissolution of the sample material in the solvent, wherein thefirst time at which the solution is sampled to obtain the first aliquotis within about 1 minute after the initiation time, t_(o), the samplingfrequencies defined by the difference in time between the first time andthe second time, and by the difference in time between the second timeand the third time, are each not more than about 1 minute.
 9. The methodof claim 8 wherein the solution is sampled at least six times during thedissolution period, the method further comprising sampling a portion ofthe solution at a fourth time within the dissolution period to obtain afourth aliquot of the solution, subsampling a portion of the fourthaliquot of the solution to obtain a fourth sub-aliquot of the solution,and determining the concentration of the sample material in the fourthsub-aliquot of the solution, sampling a portion of the solution at afifth time within the dissolution period to obtain a fifth aliquot ofthe solution, subsampling a portion of the fifth aliquot of the solutionto obtain a fifth sub-aliquot of the solution, and determining theconcentration of the sample material in the fifth sub-aliquot of thesolution, sampling a portion of the solution at a sixth time within thedissolution period to obtain a sixth aliquot of the solution,subsampling a portion of the sixth aliquot of the solution to obtain asixth sub-aliquot of the solution, and determining the concentration ofthe sample material in the sixth sub-aliquot of the solution, whereinthe fourth time is after the third time, the fifth time is after thefourth time, and the sixth time is after the fifth time.
 10. The methodof claim 9 wherein the sampling frequencies defined by the difference intime between the fourth time and the fifth time, and by the differencein time between the fifth time and the sixth time, are each not morethan about 1 minute.
 11. The method of claim 10 wherein the solution issampled at least six times over at least two distinct sampling intervalsduring the dissolution period, a first sampling interval comprising thefirst time, the second time and the third time, and a second samplinginterval comprising the fourth time, the fifth time and the sixth time,the first and second sampling intervals being separated in time by anon-sampling interval during which no sampling of the solution occurs,the non-sampling interval being defined by a period of time having aduration not less than about two times the longest of the samplingfrequencies.
 12. The method of claim 1 wherein the sampling frequencydefined by the difference in time between the first time and the secondtime is not more than about 1 minute.
 13. The method of claim 1 furthercomprising combining the sample material with the solvent at aninitiation time, t₀, to initiate dissolution of the sample material inthe solvent, wherein the first time at which the solution is sampled toobtain the first aliquot is within about 1 minute after the initiationtime, t_(o).
 14. The method of claim 13 wherein the first time at whichthe solution is sampled to obtain the first aliquot is within about 30seconds after the initiation time, t_(o).
 15. The method of claim 13wherein the first time at which the solution is sampled to obtain thefirst aliquot is within about 10 seconds after the initiation time,t_(o).
 16. The method of claim 1 wherein the solution is sampled usingan automated sampling probe, the sampling probe having a distal end anda proximate end, the distal end of the sampling probe being positionablein fluid communication with the solution in the sample container forsampling a portion of the solution to obtain sampled aliquots, theproximate end of the sampling probe being in fluid communication througha flow path with a sub-sampling device for obtaining the sub-sampledsub-aliquots, the method further comprises filtering the first aliquotof the solution during or after sampling to obtain a filtered firstaliquot of the solution, and filtering the second aliquot of thesolution during or after sampling to obtain a filtered second aliquot ofthe solution, the sampled aliquots being filtered with an in-line filterintegral with the sampling probe or in fluid communication with theproximate end of the sampling probe in the flow path providing fluidcommunication to the sub-sampling device, the remainder portion of thefirst sub-aliquot and the remainder portion of the second sub-aliquotare returned from the sub-sampling device to the solution through theflow path and the sampling probe in a reverse flow direction, and theremainder portions of the sub-aliquots backflush the in-line filterwhile being returned to the solution in the reverse flow direction. 17.The method of claim 1 wherein the sample material is a solid.
 18. Themethod of claim 17 further comprising combining the solid samplematerial with the solvent to initiate dissolution and to form adispersion comprising the solution and undissolved solid sample materialdispersed in the solution.
 19. The method of claim 18 wherein thedispersion is a substantially uniform dispersion.
 20. The method ofclaim 1 wherein the sample material comprises a drug candidate.
 21. Themethod of claim 20 wherein the sample material consists essentially of adrug candidate.
 22. The method of claim 22 wherein the sample materialcomprises a drug composition comprising one or more drug candidates andone or more excipients.
 23. The method of claim 1 further comprisingcombining not more than about 50 mg of the sample material with thesolvent to initiate dissolution of the sample material in the solvent.24. The method of claim 23 wherein not more than tout 20 mg of thesample material is combined with the solvent.
 25. The method of claim 23wherein not more than about 10 mg of the sample material is combinedwith the solvent.
 26. The method of claim 23 wherein not more than about5 mg of the sample material is combined with the solvent.
 27. The methodof claim 23 wherein not more than tout 2 mg of the sample material iscombined with the solvent.
 28. The method of claim 23 wherein not morethan about 1 mg of the sample material is combined with the solvent. 29.The method of claim 23 wherein not more than about 0.5 mg of the samplematerial is combined with the solvent.
 30. The method of claim 23wherein the sample material is combined with an amount of solventeffective for forming a solution having a detectable concentration ofsample material in the solution.
 31. The method of claim 23 wherein thesample material is combined with an amount of solvent effective forforming solution having a concentration of sample material in thesolution ranging from about 0.01 mg/ml to about 10 mg/ml.
 32. The methodof claim 23 wherein an amount of sample material ranging from 0.01 mg to0.5 mg is combined with an amount of solvent ranging from 1 ml to 10 ml.33. The method of claim 1 wherein the surface area of the samplematerial is known or determined, the method further comprisingdetermining an intrinsic dissolution rate for the sample material forone or more times during the dissolution period.
 34. The method of claim1 wherein the sample material is a first sample material, the methodfurther comprising repeating each step of the method for at least oneother distinct second sample material, and comparing a relativedissolution rate for the first sample material and the second samplematerial for one or more times during the dissolution period.
 35. Themethod of claim 1 further comprising generating a data set that definesthe dissolution profile, the data set being generated by a protocol thatcomprises defining a first data point of the dissolution profile byassociating the determined concentration of sample material in the firstaliquot with the first time, and defining a second data point of thedissolution profile by associating the determined concentration ofsample material in the second aliquot with the second time.
 36. Themethod of claim 1 wherein the sample material is a one member of anarray of sample materials, the method further comprising repeating eachstep of the method for at least one other member of the array.
 37. Themethod of claim 36 wherein the array of sample materials comprises fouror more sample materials, the method further comprising for each of thefour or more sample materials, combining the sample material with thesolvent in an individual, separate container, the container for each ofthe four or more sample materials being selected from four or morecontainers that are formed in or supported on a common substrate. 38.The method of claim 36 wherein for each of the four or more samplematerials, at least one of the sample material or the solvent areprovided to the container by an automated dispensing probe at theinitiation time, t_(o), and the solution is sampled at the first time toobtain the first aliquot using an automated sampling probe.
 39. Themethod of claim 36 wherein the array of sample materials comprises fouror more sample materials, each of the four or more sample materialshaving a chemical composition that is different from each other.
 40. Themethod of claim 36 wherein the array of sample materials comprises twoor more sample materials, each of the two or more sample materialshaving a crystalline structure different from each other.
 41. The methodof claim 36 wherein the array of sample materials comprises two or moresample materials, each of the two or more sample materials having thesame chemical composition and having a crystalline structure differentfrom each other.
 42. The method of claim 36 wherein the ray of samplematerials comprises four or more sample materials that are substantiallythe same, the method further comprising combining each of the four ormore sample materials with a different dissolution environment.
 43. Amethod for determining a dissolution profile for a sample material, themethod comprising dissolving at least a portion of the sample materialin a solvent over a period of time to form a solution, the period oftime defining a dissolution period, the solution having a concentrationof the sample material that varies during the dissolution period, thedissolving including combining the sample material with the solvent in acontainer to initiate dissolution of the sample material in the solvent,at least one of the sample material or the solvent being provided to thecontainer by an automated dispensing probe at an initiation time, t_(o),using an automated sampling probe for sampling the solution at leasttwice during the dissolution period, the solution being sampled at afirst time within the dissolution period to obtain a first aliquot ofthe solution, and at a later second time within the dissolution periodto obtain a second aliquot of the solution, subsampling a portion of thefirst aliqout of the solution to obtain a first sub-aliquot of thesolution, determining the concentration of the sample material in thefirst sub-aliquot of the solution, subsampling a portion of the secondaliqout of the solution to obtain a second sub-aliquot of the solution,and determining the concentration of the sample material in the secondsub-aliquot of the solution.
 44. The method of claim 43 furthercomprising controlling the dispensing probe independently of thesampling probe.
 45. A method for determining a dissolution profile for asample material, the method comprising dissolving at least a portion ofthe sample material in a solvent over a period of time to form asolution, the period of time defining a dissolution period, the solutionhaving a concentration of the sample material that varies during thedissolution period, sampling a portion of the solution at least twiceduring the dissolution period, the solution being sampled at a firsttime within the dissolution period to obtain a first aliquot of thesolution, and at a later second time within the dissolution period toobtain a second aliquot of the solution, including filtering the firstaliquot of the solution during or after sampling to obtain a filteredfirst aliquot of the solution, and filtering the second aliquot of thesolution during or after sampling to obtain a filtered second aliquot ofthe solution, subsampling a portion of the first aliquot of the solutionto obtain a first sub-aliquot of the solution, determining theconcentration of the sample material in the first sub-aliquot of thesolution, subsampling a portion of the second aliquot of the solution toobtain a second sub-aliquot of the solution, and determining theconcentration of the sample material in the second sub-aliquot of thesolution.
 46. The method of claim 45 wherein the solution is sampledusing an automated sampling probe, the sampling probe having a distalend and a proximate end, the distal end of the sampling probe beingpositionable in fluid communication with the solution in the samplecontainer for sampling a portion of the solution to obtain sampledaliquots, and the sampled aliquots are filtered with an in-line filterintegral with the sampling probe or in fluid communication with theproximate end of the sampling probe.
 47. A method for determining adissolution profile for a sample material, the method comprisingdissolving at least a portion of the sample material in a solvent over aperiod of time to form a solution, the period of time defining adissolution period, the solution having a concentration of the samplematerial that varies during the dissolution period, the dissolvingincluding varying a property of the solvent over time during thedissolution period, sampling a portion of the solution at least twiceduring the dissolution period, the solution being sampled at a firsttime within the dissolution period to obtain a first alipuot of thesolution, and at a later second time within the dissolution period toobtain a second aliquot of the solution, subsampling a portion of thefirst aliquot of the solution to obtain a first sub-aliquot of thesolution, determining the concentration of the sample material in thefirst sub-aliquot of the solution, subsampling a portion of the secondaliquot of the solution to obtain a second sub-aliquot of the solution,and determining the concentration of the sample material in the secondsub-aliquot of the solution.
 48. The method of claim 47 furthercomprising varying the chemical composition of the solvent over timeduring the dissolution period.
 49. The method of claim 47 furthercomprising varying the pH of the solvent over time during thedissolution period.
 50. The method of claim 47 further comprisingvarying the temperature of the solvent over time during the dissolutionperiod.
 51. A method for generating data defining a dissolution profilefor a sample material, the method comprising dissolving at least aportion of the sample material in a solvent over a period of time toform a solution, the period of time defining a dissolution period, thesolution having a concentration of the sample material that variesduring the dissolution period, sampling a portion of the solution at afirst time within the dissolution period to obtain a first aliquot ofthe solution, subsampling a portion of the first aliquot of the solutionto obtain a first sub-aliquot of the solution, such subsampling therebyalso resulting in a remainder portion of the first aliquot, returningthe remainder portion of the first aliquot to the solution, sampling aportion of the solution at a later second time within the dissolutionperiod after the remainder portion of the first aliquot is returned tothe solution, to obtain a second aliquot of the solution, subsampling aportion of the second aliquot of the solution to obtain a secondsub-aliquot of the solution, such subsampling thereby also resulting ina remainder portion of the second aliquot, returning the remainderportion of the second aliquot to the solution, determining theconcentration of the sample material in the first sub-aliquot of thesolution, whereby a first data point of the dissolution profile isdefined by association of the concentration determined in the firstsub-aliquot with the first time, and determining the concentration ofthe sample material in the second sub-aliquot of the solution, whereby asecond data point of the dissolution profile is defined by associationof the concentration determined in the second sub-aliquot with thesecond time.
 52. The method of claim 51 further comprising providing afirst make-up aliquot of a liquid media to the solution, the firstmake-up aliquot having a volume about the same as the volume of thefirst sub-aliquot, the first make-up aliquot being provided aftersampling to obtain the first aliquot of the solution, and beforesampling to obtain the second aliquot of the solution, and providing asecond make-up aliquot of the liquid media to the solution, the secondmake-up aliquot having a volume about the same as the volume of thesecond sub-aliquot, the second make-up aliquot being provided aftersampling to obtain the second aliquot of the solution.
 53. A system fordetermining a dissolution profile for a sample material, the systemcomprising a sample container for dissolving at least a portion of thesample material in a solvent over a dissolution period of time to form asolution that has a concentration of the sample material that variesduring the dissolution period, an automated sampling probe for samplinga portion of the solution at least twice during the dissolution period,the solution being sampled at first and second times within thedissolution period to obtain fist and second aliquots of the solution,respectively, the sampling probe having a distal end positionable influid communication with the solution in the sample container, andhaving a proximate end, a sub-sampling device including a sampling valvein fluid communication with the proximate end of the sampling probe, thesub-sampling device being configured for subsampling a portion of eachof the first and second aliquots of the solution to obtain first andsecond sub-aliquots of the solution, and an analytical unit fordetermining the concentration of the sample material in the first andsecond sub-aliquots of the solution.
 54. The system of claim 53 furthercomprising an automated dispensing probe for providing the samplematerial to the container.
 55. The system of claim 53 further comprisingan automated dispensing probe for providing the sample material to thecontainer, and a control system for independently controlling theautomated sampling probe and the automated dispensing probe.
 56. Thesystem of claim 53 further comprising a microprocessor having automationsoftware for controlling the automated sampling probe and thesub-sampling device.
 57. The system of claim 53 further comprising anin-line filter for filtering the first and second aliquots of thesolution during or after sampling to obtain filtered first and secondaliquots of the solution, respectively, the in-line filter beingintegral with the sampling probe or in fluid communication with theproximate end of the sampling probe in the flow path providing fluidcommunication to the sub-sampling device.
 58. The system of claim 53wherein the automated sampling probe is a component of an automatedliquid handling system, the liquid-handling system further comprising arobotic arm for translating the automated sampling probe, and a pump incontinuous or selectable fluid communication with the sampling probe forproviding a motive force for withdrawing a portion of the solution intothe probe to effect sampling.
 59. The system of claim 58 wherein thepump is a positive displacement pump.
 60. The system of claim 58 whereinthe pump is a reciprocating pump.
 61. The system of claim 58 wherein thepump is a syringe pump.
 62. The system of claim 58 wherein the pump isin fluid communication with the sampling valve such that the samplingvalve provides continuous or selectable fluid communication between thepump and the proximate end of the sampling probe, and the analyticalunit comprises a detector in fluid communication with the samplingvalve.
 63. The system of claim 62 wherein the sampling valve comprises asample loop, and the sampling valve is configured so that in a firstselectable position, the pump is in fluid communication with theproximate end of the sampling probe through a flow path that includesthe sample loop, and so that in a second selectable position, the pumpis in fluid communication with the proximate end of the sampling probethrough a flow path that bypasses the sample loop.
 64. The system ofclaim 63 further comprising an in-line filter for filtering the firstand second aliquots of the solution during or after sampling to obtainfiltered first and second aliquots of the solution, respectively, thein-line filter being integral with the sampling probe or in fluidcommunication with the proximate end of the sampling probe in the flowpath providing fluid communication to the sampling valve, such that whenthe sampling valve is in the first selectable position, at least part ofthe filtered sampled aliquot is loaded into the sample loop, and suchthat when the sampling valve is in the second selectable position, aremainder portion of the filtered sampled aliquot not loaded into thesample loop is returned to the solution in a reverse flow directionthrough the flow path that bypasses the sample loop and backflushes thein-line filter.
 65. The system of claim 62 wherein the sampling valvecomprises a sample loop, and the sampling valve is configured so that ina first selectable position, the sample loop is selectably aligned to asampling flow pat for loading at least part of the sampled aliquot intothe sample loop, and so that in a second selectable position, the sampleloop is selectably aligned to a detection flow path for discharging thecontents the sample loop as a sub-aliquot of the sampled aliquot to thedetector.
 66. The system of claim 62 wherein the pump is a first pumpand the sampling valve is a multi-port sampling valve comprising asample loop, the sampling valve being configured in a first selectableposition for loading at least part of the sampled aliquot into thesample loop through a sampling flow path from the proximate end ofsampling probe to the first pump, the sampling valve being configured ina second selectable position for returning a remainder portion of thesampled aliquot not loaded into the sample loop to the container througha remainder-return flow path from the first pump to the proximate end ofthe sampling probe, and the sampling valve being further configured inone of the selectable positions for discharging the contents the sampleloop as a sub-aliquot of the sampled aliquot through a detection flowpath from a second pump to the detector.
 67. The system of claim 62wherein the pump is a first pump and the sampling valve is a multi-portsampling valve comprising a first sample loop and a second sample loop,the sampling valve being configured in a first selectable position forloading at least part of the sampled aliquot into the first sample loopthrough a sampling flow path from the proximate end of sampling probe tothe first pump, the sampling valve being configured in the secondselectable position for returning a remainder portion of the sampledaliquot not loaded into the first sample loop to the container through aremainder-return flow path from the first pump to the proximate end ofthe sampling probe, the sampling valve being further configured in thesecond selectable position for loading a make-up liquid media into thefirst sample loop and simultaneously advancing the contents of the firstsample loop to a second sample loop, the sampling valve being furtherconfigured in the first selectable position for discharging the contentsthe second sample loop as a sub-aliquot of the sampled aliquot through adetection flow path from a second pump to the detector, the samplingvalve being further configured in the first selectable position forproviding the make-up liquid media loaded into the first sample loop asa make-up aliquot through a make-up flow path from a third pump to theproximate end of the sampling probe for delivery to the solution in thecontainer.
 68. The system of claim 62 wherein the analytical unitfurther comprises a separation device in fluid communication with thesampling valve and with the detector, the separation device beingadapted for separating one or more components of the subsampledsub-aliquots discharged from the sampling valve from other componentsthereof, the detector being adapted for detecting a property of the oneor more separated components.
 69. The system of claim 68 wherein theseparation device is a liquid chromatography column.
 70. The system ofclaim 68 wherein the separation device is a liquid chromatographycolumn, and the detector is selected from one or more of alight-scattering detector, a refractive index detector, a fluorescencedetector, an infrared detector, and a spectroscopic detector.
 71. Thesystem of claims 53 or 62 wherein the analytical unit comprises a flowdetector in fluid communication with the sub-sampling device.
 72. Thesystem of claims 53 or 62 wherein the analytical unit further comprisesa separation device in fluid communication with the sub-sampling device.73. The system of claims 53 or 62 wherein the analytical unit comprisesa liquid chromatography system or a flow-injection analysis system.